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Mesangial Cell-Binding Anti-DNA Antibodies in Patients with Systemic Lupus Erythematosus

Division of Nephrology, Department of Medicine, the University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China.

Correspondence to: Professor Tak-Mao Chan, Department of Medicine, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China. Phone: 852-2855-4041; Fax: 852-2872-5828; E-mail: dtmchan{at}hkucc.hku.hk

	Abstract

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ABSTRACT. The mechanisms by which anti-DNA antibodies contribute to the pathogenesis of lupus nephritis (LN) remain to be elucidated. This study investigates the binding of polyclonal anti-DNA immunoglobulins from patients with systemic lupus erythematosus (SLE) to human mesangial cells (HMC) in vitro. Testing of cross-sectional serum samples from 280 LN patients (108 during active disease; 172 during remission), 35 SLE patients without renal involvement, 72 patients with non-lupus primary glomerular diseases, and 37 healthy subjects with a cellular enzyme-linked immunosorbent assay showed significant IgG mesangial cell-binding activity in patients with SLE, particularly those with active LN (P < 0.0001). Significant HMC-binding activity was demonstrated in 83.9%, 42.8%, and 47.1% of patients with active LN, inactive LN, and non-renal SLE, respectively. This was predominantly attributed to binding by anti-DNA antibodies, and immune complex binding accounted for 4.6%, 3.5%, and 2.8% of seropositive samples in the respective groups. Longitudinal studies in 27 LN patients demonstrated correlation between serial levels of anti-DNA antibodies, serum HMC-binding activity, and disease activity in 18 patients (66.7%). Affinity-purified polyclonal IgG anti-DNA antibodies from sera with HMC-binding activity showed significant binding to cultured HMC, and to a lesser extent glomerular and proximal tubular epithelial cells and human umbilical vein endothelial cells, but not tumor cell lines, peritoneal mesothelial cells, bronchial epithelial cells, or fibroblasts. The binding of anti-DNA antibodies to HMC was increased 1.47-fold (P = 0.0059) after the removal of Ig-associated DNA by DNase treatment, but it was unaffected by DNase treatment of HMC membrane. Controlled trypsinization of membrane proteins in HMC resulted in a 1.26-fold (P = 0.0025) increase in their binding by anti-DNA antibodies. In conclusion, subsets of anti-DNA antibodies from patients with SLE are capable of binding to HMC. The association of such binding with renal involvement and disease activity and its modulation by DNA concentration suggest that Ig binding to HMC can be a potential marker for disease activity in selected patients and that the binding of anti-DNA antibodies to HMC may be a pathogenetic mechanism in LN.

	Introduction

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Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the presence of autoantibodies against nuclear antigens (1). Lupus nephritis (LN) is a frequent and severe organ manifestation in which mesangial proliferation and deposition of immunoglobulins and complement components within the mesangium are prominent features (2). Anti-DNA antibodies have been demonstrated in renal and glomerular eluates obtained from SLE patients and lupus mice (3–5). The correlation between circulating anti-DNA antibodies and disease activity presents further evidence that these antibodies participate in pathogenetic mechanisms. Furthermore, glomerulonephritis could be induced in non-autoimmune mice upon inoculation with the transgene that encodes the secreted form of an IgG anti-DNA antibody, indicating that anti-DNA antibodies are instrumental in the initiation of LN (6).

However, the role of anti-DNA antibodies in the pathogenetic mechanisms of LN has not been fully elucidated (7,8), and the heterogeneity of these antibodies implies variable pathogenetic involvement. Characteristics of anti-DNA antibodies that have been associated with pathogenicity include an IgG isotype, cationic charge, high affinity for binding to double-stranded DNA, and cross-reactivity with different glomerular components, such as laminin, collagen, and heparan sulfate proteoglycans (8–14). Glomerular binding by autoantibodies has been associated with pathogenicity and disease activity (15–17). It remains unclear how these antibodies interact with resident cells or components in the glomerulus.

The strategic location of mesangial cells in the glomerulus, juxtaposed to adjacent capillary loops, facilitates their interaction with immune deposits, complement components, and inflammatory mediators (18). Mesangial cell proliferation and the deposition of anti-DNA antibodies in the mesangium are characteristic features in LN (2). It is thus pertinent to examine the mechanisms by which anti-DNA antibodies interact with mesangial cells and the pathogenetic consequences of such interactions. We have investigated the binding of immunoglobulins from patients with SLE to human mesangial cell (HMC) in vitro. Our results showed that anti-DNA antibodies accounted for the serum HMC-binding activity. The latter also correlated with clinical activity in selected patients. The binding of anti-DNA antibodies to HMC could be modulated by Ig-associated DNA and was mediated in part by membrane proteins.

	Materials and Methods

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All chemicals were purchased from Sigma (China South, Hong Kong) unless otherwise stated and were of the highest purity. Tissue culture flasks were purchased from Falcon (Becton Dickinson, Hong Kong), and culture media from Life Technologies (Hong Kong).

Serum Samples from Patients with LN
For the cross-sectional study to determine the prevalence of HMC-binding immunoglobulins, 280 sera from 280 patients (222 women, 58 men; mean age, 45.7 ± 14.9 yr) with a history of biopsy-proven diffuse or severe focal proliferative LN and who satisfied four or more of the American Rheumatism Association criteria for SLE (19) were included. These serum samples were classified as either "active" or "inactive" on the basis of both clinical and serologic assessment. Active disease was associated with an SLE Disease Activity Index (SLEDAI) 10, while remission samples were obtained when the SLEDAI was 4 (20). Serum samples from SLE patients without renal involvement (n = 35), patients with non-lupus primary glomerular diseases (n = 72; thin membrane disease, 7; membrano-proliferative glomerulonephritis, 16; membranous nephropathy, 16; focal segmental glomerulosclerosis, 15; minimal change disease, 18), and healthy subjects (n = 37) were included for comparison. In addition, multiple serial serum samples were obtained from 27 patients with diffuse proliferative LN (19 women, 8 men; mean age, 35.1 ± 11.5 yr) over 4.5 ± 1.2 yr to study the association between Ig binding to HMC, the level of anti-DNA antibodies, and clinical disease activity.

Cell Culture
HMC were cultured from nephrectomized kidneys according to established methods (21). Briefly, cells derived from collagenase-treated glomeruli were maintained in RPMI 1640 medium supplemented with 20% (vol/vol) fetal calf serum (FCS), glutamine (2 mM), transferrin (5 µg/ml), insulin (5 µg/ml), sodium selenite (5 ng/ml), penicillin (100 IU/ml), and streptomycin (100 µg/ml). HMC were characterized by their stellate morphology, ability to form hillocks, and immunohistochemical staining (positive for vimentin; negative for cytokeratin and von Willebrand Factor). HMC were passaged at a split ratio of 1:3, and growth-arrested cells of the 4^th through 7^th passage were used in experiments. These cells showed no significant variation in viability, proliferation rate, cellular protein concentration (/10⁵ cells), and the expression of smooth muscle actin, vimentin, or extracellular matrix synthesis (data not shown).

Glomerular epithelial cells (GEC) were isolated from the supernatant of collagenase-treated glomeruli (22). Human peritoneal mesothelial cells (HPMC) were isolated from normal omental specimens according to established protocol (23). Proximal tubular epithelial cells (PTEC), human umbilical vein endothelial cells (HUVEC), human pleural mesothelial cells (MeT-5A), and human bronchial epithelial cells (NHBE) were purchased from Clonetics (Advanced Tech and Ind Comp Ltd, Hong Kong); CHO cells and 3T3 Swiss fibroblasts were kind gifts from Dr. Nancie Chen (University of Hong Kong, Hong Kong).

Determination of Cellular Protein Concentration
Confluent growth-arrested cells (HMC, GEC, PTEC, HUVEC, HPMC, MeT-5A, NHBE, CHO cells, and 3T3 Swiss fibroblasts) cultured in triplicate in 96-well plates were lysed with 4 M urea buffer, 20 mM sodium acetate, pH 6.0, containing 1% (vol/vol) Triton X-100 (50 µl). The protein concentration in each sample was determined using a modified Lowry assay according to the manufacturer’s instructions (Bio-Rad, Hong Kong).

Removal of Immune Complexes (IC) from Serum Samples
Two approaches were used to remove IC in serum samples from patients with SLE.

Precipitation of IC with polyethylene glycol (PEG).
IC in sera (0.5 ml) were precipitated with PEG (3.4% [wt/vol] final concentration) for 30 min at 4°C. Samples were centrifuged at 15,000 g for 30 min to separate the IC-free sera from the precipitate.

Removal of IC by Protein G-Sepharose.
Sera (5 µl) diluted in phosphate-buffered saline (PBS; 80 µl) were incubated with protein G-Sepharose beads (10 µl, Amersham Pharmacia Biotech, Hong Kong) for 15 min at room temperature with constant agitation before centrifugation to remove the protein G-Sepharose-bound IC (24).

HMC-binding activity was compared between serum samples with or without the removal of IC by the above procedures.

Cellular Enzyme-Linked Immunosorbant Assay (ELISA) to Measure HMC Binding by Either Serum Ig or Polyclonal Anti-DNA Antibodies
HMC were seeded into 96-well tissue culture plates at 20,000 cells/well in RPMI 1640 medium containing 20% FCS. 90% confluent cells were depleted of FCS for 72 h before being used in experiments. Measurement of lactate dehydrogenase (LDH) release confirmed maintenance of cell viability (5.12 ± 2.14% versus 6.01 ± 1.32% cytotoxicity before and after serum starvation, respectively; P = 0.147; n = 6). HMC were fixed with 1% (wt/vol) paraformaldehyde in PBS (pH 7.5) for 5 min at room temperature. The cells were washed thrice with PBS between incubations, and all incubations were for 1 h at 37°C unless otherwise stated. HMC were blocked with 3% (wt/vol) bovine serum albumin (BSA) followed by normal human IgG (100 µg/ml) to block Fc receptor-mediated binding. HMC were subsequently incubated with 100 µl of test sera (dilution 1:100) or anti-DNA preparations (final IgG concentration, 10 µg/ml). In some experiments, increasing concentrations of DNA (0 to 10 µg/ml) were added to anti-DNA antibodies for 2 h at 37°C before incubation with HMC to assess competition for the binding antigen on the cell surface. HMC were then incubated with goat anti-human IgG F(ab) conjugated with alkaline phosphatase (5 µg/ml; Biosource Int, Hong Kong). Goat anti-human IgA or IgM F(ab) was used as the second antibody in experiments to assess binding by IgA or IgM immunoglobulins. Cross-reactivity of anti-human IgG, IgA, and IgM antibodies toward other isoforms were 8.2 ± 3.5%, 5.2 ± 3.6%, and 6.1 ± 3.1%, respectively. Ig binding to HMC was detected by incubation with para-nitrophenol phosphate for 45 min at room temperature, and optical density (OD) was measured at wavelength A_405/420 on a Titreteck Multiscan MCC/340 spectrophotometer (Bio-Rad, Hong Kong) when the positive control showed an OD of 1.5. Pooled serum or polyclonal anti-DNA antibodies from a patient who demonstrated persistent high HMC-binding activity due to anti-DNA antibodies were used as positive controls in respective experiments. Intra-assay and inter-assay coefficients of variance were 4.5 ± 0.7% and 5.3 ± 0.6%, respectively. A standard curve was obtained by plotting mean OD against mean IgG bound to HMC (the latter determined by the difference in IgG concentration of the tested sample before and after incubation with HMC) from 20 LN patients who showed variable degrees of HMC-binding activity. This standard curve (correlation coefficient = 0.97) was subsequently used to determine the amount of IgG binding to HMC in control and tested samples. The amount of IgG bound to HMC was expressed as µg of bound IgG/µg of cell protein. Seropositivity for HMC-binding of any tested sample was denoted by results that exceeded mean + 3 SD of the corresponding specimens from healthy subjects.

Cellular ELISA with GEC, PTEC, HUVEC, HPMC, MeT-5A, NHBE, CHO cells, and 3T3 Swiss fibroblasts as substrate were also performed to examine the cellular specificity of Ig binding.

Isolation of Polyclonal Anti-DNA Antibodies from SLE Sera
This was achieved through sequential affinity chromatography (25). Aliquots (0.5 ml) of serum samples were loaded onto native DNA-cellulose column (Amersham Pharmacia Biotech) equilibrated with 25 mM Tris buffer (pH 8.0) at a flow rate of 0.5 ml/min. Non-DNA-binding fractions were flushed with the above buffer, and anti-DNA antibodies were eluted with a linear NaCl gradient. The absorbance at A₂₈₀ was measured continuously throughout the purification process. The columns were subsequently washed with 20 mM Tris-HCl (pH 7.4) containing 2 M NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM -mercaptoethanol before further elution. This did not yield additional immunoglobulins. Control experiments were performed by passage of serum samples through a column of microgranular cellulose in the absence of immobilized DNA. Anti-DNA antibodies of the IgG class were isolated by protein A Sepharose affinity chromatography (25,26). Isolated anti-DNA and non-DNA-binding samples were desalted and concentrated 20-fold using Ultrafree-4 centrifugal filter units (Millipore Asia, Hong Kong) before determination of IgG concentration, anti-DNA activity, or use in experiments. The purity of eluted IgG was confirmed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Flow Cytometry to Examine Ig Binding to HMC
Confluent HMC were treated with 0.05% (wt/vol) trypsin with 0.02% (wt/vol) EDTA for 5 min at 37°C and then neutralized with trypsin inhibitor (1 mg/ml). HMC were then cultured in suspension for 4 h, pelleted by centrifugation at 1500 g for 5 min, washed thrice with PBS, and incubated with serum samples, anti-DNA antibodies, or control IgG (10 µg/ml) for 30 min at 4°C in Krebs-Ringer bicarbonate buffer containing 1% BSA. Unbound immunoglobulins were washed with the above buffer. HMC were then incubated with goat anti-human IgG F(ab) conjugated with FITC (final concentration, 5 µg/ml) at 4°C for 1 h, washed thrice with PBS, and resuspended in 0.5 ml of PBS containing 0.5% (vol/vol) formaldehyde. Ig binding was analyzed by flow cytometry (Coulter Epics XL Flow Cytometer, Coulter, FL), with 10,000 cells counted for each sample. Analysis was performed with XL System II software.

Measurement of Anti-DNA Activity in Serum and Isolated Anti-DNA Fractions
This was measured using a commercial ELISA (Microplate autoimmune anti-DNA quantitative ELISA, Bio-Rad, Hong Kong). Samples giving a value >60 IU/ml were considered positive. The lower and upper limits of detection for this assay were 20 IU/ml and 1000 IU/ml, respectively.

Measurement of IgG, IgA, or IgM Concentration in Anti-DNA Antibody Preparations
IgG, IgA, and IgM concentration in anti-DNA antibody preparations were measured by ELISA. Goat anti-human IgG, IgA, or IgM (10 µg/ml, 200 µl; Biosource International, Hong Kong) in 0.05 M carbonate buffer (pH 9.5) were coated onto 96-well microtiter plates (Immulon 2; Dynex Technologies, Hong Kong) at 4°C overnight. After washing thrice with PBS/0.1% (vol/vol) Tween-20 (PBST), the plates were blocked with 3% (wt/vol) BSA in PBS for 1 h at 37°C. After washing with PBS, samples (starting dilution, 1:1000) or standards (range, 0 to 500 ng/ml; ICN Biomedicals, Eschwege, Germany) were added in duplicate in serial dilutions and incubated for 1 h at 37°C. After washing in PBS, the relevant alkaline phosphatase-conjugated goat anti-human Ig (5 µg/ml; Biosource, Hong Kong) was added and incubated for 1 h at 37°C. Bound immunoglobulins were detected by the addition of para-nitrophenol phosphate, and the absorbance was read at A_405/420. Prior experiments have demonstrated <5% isoform cross-reactivity of the antibodies. Intra-assay and inter-assay coefficients of variance were 3.5 ± 0.3% and 4.9 ± 0.4%, respectively, for IgG, 2.1 ± 1.3% and 6.2 ± 2.4%, respectively, for IgA, and 4.3 ± 2.7% and 7.1 ± 0.2%, respectively, for IgM.

Digestion of DNA with DNase I
Removal of DNA from Anti-DNA Antibodies.
Sera or anti-DNA antibodies (final IgG concentration, 10 µg/ml) were incubated with DNase I (40 µg/ml) and MgCl₂ (10 mM) for 1 h at 37°C, and the reaction was stopped by the addition of EDTA (15 mM) (27).

Removal of DNA from the Surface of HMC.
DNA was removed from HMC surface after fixation with 1% (wt/vol) paraformaldehyde. After washing with PBS, the cells were incubated with 25 µg/ml DNase I in PBS containing 10 mM MgCl₂ for 1 h at 37°C, after which the reaction was stopped by the addition of EDTA (15 mM). Prior experiments had confirmed that DNase I at 25 µg/ml removed cell surface DNA maximally (determined by agarose gel electrophoresis and ethidium bromide staining of isolated plasma membrane from HMC before and after DNase treatment) without causing cell detachment or cytotoxicity (assessed in parallel experiments by phase contrast microscopy and LDH release respectively).

Removal of Transmembrane Proteins from HMC by Controlled Trypsin Digestion
Fixed HMC were treated with 10 µg/ml trypsin (tissue culture grade) in PBS for 10 min at 4°C before neutralization with trypsin inhibitor (1 mg/ml) in PBS. The cells were washed thrice with PBS. This concentration of trypsin resulted in maximal release of [³⁵S]-methionine-labeled transmembrane proteins without affecting cell attachment (data not shown).

Immunohistochemical Staining
HMC were seeded onto glass coverslips at a density of 10,000 cells/cm² and cultured for 48 h before fixing with 3.5% (wt/vol) paraformaldehyde in PBS for 10 min at room temperature. Half of the slides were incubated with trypsin (10 µg/ml) for 10 min at 4°C before staining. Cells were then incubated with anti-DNA antibodies or normal human IgG controls (final IgG concentration, 10 µg/ml) for 1 h at 37°C, washed thrice with PBS, and incubated with second antibody conjugated with FITC for 1 h at 37°C in a darkened humidified chamber. Cells were washed in PBS and mounted in fluorescence mounting medium (DAKO, Gene Company Ltd, Hong Kong), and epifluorescence was monitored using a Zeiss Axiovert 135 microscope (Carl Zeiss, Gold Pacific Ltd, Hong Kong). Photographs were taken on Kodak Max 400 film (Eastman Kodak, Rochester, NY). The localization of antibody staining was further confirmed using confocal microscopy (Aviovert 135M and Microsystems LSM, Carl Zeiss).

Statistical Analyses
All experiments were repeated at least three times using HMC from three separate donors. Results are expressed as mean ± SD. Statistical analyses were performed using InStat GraphPad software (GraphPad, San Diego, CA). Comparison between active and inactive samples was performed by the Mann-Whitney U test. Anti-DNA antibody binding to HMC was compared with that of non-anti-DNA Ig fractions from the same patient by the Wilcoxon signed-ranks test. Correlation between HMC-binding IgG and the level of anti-DNA IgG antibodies was examined with Spearman’s method. Two-tailed P < 0.05 was considered statistically significant.

	Results

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HMC-Binding Immunoglobulins in Patients with SLE
Preliminary studies using 20 serum samples with positive HMC-binding activity showed similar HMC-binding results for paraformaldehyde-fixed and live HMC (4.2 ± 1.2 µg of bound IgG/µg of cell protein and 4.0 ± 1.5 µg of bound IgG/µg of cell protein respectively; P = 0.1703). Fixed cells were used in subsequent cellular ELISA.

Cellular ELISA with HMC, GEC, PTEC, HUVEC, HPMC, MeT-5A, NHBE, CHO cells, and 3T3 Swiss fibroblasts as substrate to examine the cellular specificity of Ig binding, demonstrated IgG binding in serum samples from patients with SLE to HMC, glomerular and tubular epithelial cells, and HUVEC, but insignificant binding to the other cell types relative to normal IgG (Figure 1). We have previously investigated the binding of anti-DNA antibodies to HUVEC (25). HMC binding by anti-DNA antibodies were investigated in this study.

View larger version (15K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Comparison of binding to different cell types by IgG from normal control sera () and IgG from serum samples of patients with lupus nephritis (LN; ). Patients with LN demonstrated significant IgG binding to human mesangial cells (HMC), glomerular epithelial cells (GEC), proximal tubular epithelial cells (PTEC), and human umbilical vein endothelial cells (HUVEC) (P < 0.001 compared with controls). The binding to HMC was significantly higher than that to GEC, PTEC, or HUVEC (P < 0.02).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. (A) Comparison of HMC-binding activity in serum samples from patients with LN, patients with systemic lupus erythematosus (SLE) but no renal involvement, patients with non-lupus primary glomerular diseases, and healthy individuals. P values refer to comparisons with healthy controls. Horizontal bars represent mean values, and open symbols represent the SD. Significant IgG binding to HMC was observed in patients with SLE, especially those with active LN. (B) HMC-binding by Ig of different isotypes in serum samples from patients with LN and healthy controls. LN sera demonstrated significant IgG, but not IgA or IgM, binding to HMC. P < 0.0001, P = 0.25, and P = 0.055 for IgG, IgA, and IgM, respectively, compared with controls.

View larger version (13K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Fluorescence Activated Cell Sorter (FACS; Becton Dickinson) analysis demonstrating significant IgG binding to HMC in serum samples from patients with LN (B and C) compared with healthy controls (A). Enhanced binding was noted during active disease (C) compared with remission samples (B). Mean fluorescent intensity for healthy controls, LN samples during remission, or active disease were 4.9%, 22.8%, and 69.3%, respectively.

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Correlation between HMC-IgG binding and anti-DNA antibody titer in patients with SLE and a history of renal involvement during active diffuse proliferative LN (•) or inactive disease ().

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. (A) Comparison of HMC binding by anti-DNA antibodies and non-DNA-binding IgG fractions isolated from patients with LN. Anti-DNA antibodies accounted for the HMC-binding immunoglobulins in all but one patient, who demonstrated significant binding to HMC in the non-anti-DNA fraction. Horizontal bars represent mean values. (B) Comparison of IgG binding to HMC between whole sera and the anti-DNA preparations isolated from the corresponding samples. HMC-binding activity was enhanced in the anti-DNA preparations. (C) Correlation between HMC binding by IgG anti-DNA antibody preparations and by IgG in the original whole serum samples.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Relationship between HMC-binding activity (•), the level of anti-DNA antibodies (), and clinical disease activity in serial serum samples of three representative patients with LN. In 66.7% of patients, HMC-binding correlated with anti-DNA antibody levels and disease activity (as shown in panel A). 29.6% of patients demonstrated positive HMC-binding activity that showed moderate temporal changes, which were less marked than those of anti-DNA antibodies (as shown in panel B). One patient (C) (3.7%) had persistent high HMC-binding despite remission.

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. (A) Effects of DNase treatment on the binding of anti-DNA antibodies to HMC. DNase treatment of anti-DNA antibodies significantly increased binding to HMC, whereas DNase treatment of cells did not have a significant effect. (B) Graph showing the effects of treating HMC with trypsin and/or DNase on their binding by DNase-treated anti-DNA antibodies. Trypsin treatment of HMC resulted in markedly enhanced binding by anti-DNA antibodies, whereas DNase treatment of HMC did not have additional effects. Horizontal bars represent mean HMC-binding by IgG.

View larger version (59K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Immunohistochemical staining to compare the binding of normal IgG (A) or IgG anti-DNA antibodies (B) to HMC. Cell surface and cytoplasmic staining was observed with anti-DNA antibodies but not with control normal IgG. Trypsinization of cell surface proteins further increased the binding by anti-DNA antibodies (C). Magnification, x400.

View larger version (79K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Immunohistochemical and confocal analyses of anti-DNA antibody staining in HMC. HMC were sectioned at 1-µm intervals beginning from the cell surface (A). HMC binding by anti-DNA antibodies was observed on the cell membrane (depicted by arrow) and within the cytoplasmic area. Magnification, x600.

	Discussion

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We observed that the HMC-binding activity in LN sera was predominantly attributable to the binding of anti-DNA antibodies to HMC. The pathogenetic significance of anti-DNA antibodies has been implicated by their correlation with disease activity (29–31) and their deposition at sites manifesting histopathologic changes (32–35). It would be of interest to speculate whether binding of anti-DNA antibodies to HMC might also contribute to the pathogenetic mechanisms of disease. In line with previous reports, we have also found that circulating anti-DNA antibodies were predominantly of the IgG isotype (36,37). Furthermore, only anti-DNA antibodies of the IgG isotype showed significant binding to HMC. Heterogeneity among the HMC-binding anti-DNA antibody populations is suggested by the observation that the relationship between IgG binding to HMC and clinical disease activity was observed in only 67% of patients.

Our findings demonstrate the binding of anti-DNA antibodies to the HMC cell surface and intracellularly, while no staining was observed within the extracellular matrix. It has been proposed that the binding of anti-DNA antibodies to glomerular structures can occur through two mechanisms: the direct binding of these antibodies to crossreactive epitopes or indirect binding via intercalating DNA and/or chromatin material (11,13,15,38–41). It has been previously reported that indirect binding was mediated by heparan sulfate or other cell surface molecules through interactions with DNA, histones, and nucleosomes (12,13,38). On the other hand, direct crossreactivity has been associated with pathogenicity (39,40,42). In this context, we observed markedly increased binding of human polyclonal anti-DNA antibodies to HMC upon DNase treatment of the antibodies, suggesting that the predominant mechanism by which these antibodies bound to HMC was through direct crossreactivity with HMC membrane epitopes. This was confirmed by the inhibition of HMC-binding when anti-DNA antibodies had been incubated with exogenous DNA. That DNase treatment of mesangial cells did not alter their binding by anti-DNA antibodies could be explained by the already insignificant amount of DNA on the cell surface. Previous studies have demonstrated preferential localization of DNA in the glomerular basement membrane but not on the cell surface in LN (43). On the other hand, the enhancement of HMC-binding activity observed with affinity isolated polyclonal anti-DNA antibodies compared to the corresponding original whole serum suggests the presence of serum factors that could inhibit the binding of anti-DNA antibodies to HMC.

Treatment of HMC with trypsin markedly increased their binding by anti-DNA antibodies. Trypsin is a serine protease with substrate specificity toward positively charged lysine and arginine side chains. Proteases are secreted by infiltrating inflammatory cells and mesangial cells in inflammatory renal diseases (44–46). Whether the synthesis of trypsin is modulated in LN remains unknown. Mesangial cells synthesize many of the cell surface or matrix components that have been demonstrated to crossreact with anti-DNA antibodies, such as collagen type IV, fibronectin, laminin, and heparan sulfate proteoglycans (47), many of which are susceptible to trypsin digestion. Thus, the modulation of anti-DNA binding to HMC by trypsin could be of pathogenetic relevance, although the nature of HMC membrane proteins that mediate the binding by anti-DNA antibodies have yet to be identified. The marked increase in anti-DNA binding upon trypsin treatment of HMC suggests that cell membrane protein(s) might have masked the binding sites for these antibodies, or that anti-DNA antibodies have higher affinity of binding to protein fragments generated after trypsin treatment. The cell membrane molecules that mediated anti-DNA binding were not likely to be associated with DNA in their normal state, because DNase treatment of the cells did not induce further changes in antibody binding. It has been reported that hyaluronan could bind anti-DNA antibodies through its negative carboxyl groups (48). Hyaluronan is a glycosaminoglycan present on the cell surface and in the extracellular matrix (49). It is devoid of a core protein and is thus resistant to trypsin digestion. Binding of hyaluronan to its cell surface receptors can reduce its binding to other molecules (49). Trypsin digestion of hyaluronan binding proteins on the cell surface may therefore unmask or enhance the capacity of hyaluronan to bind anti-DNA antibodies. Studies are being carried out to characterize the HMC membrane molecules that mediate the binding by anti-DNA antibodies.

In conclusion, we have demonstrated in patients with LN the presence of IgG anti-DNA antibodies that were capable of binding to HMC. The relationship between HMC binding by anti-DNA antibodies and disease activity and the data implicating direct crossreactivity with HMC membrane epitopes suggest that HMC-binding anti-DNA antibodies may participate in the pathogenesis of LN. Human polyclonal anti-DNA antibodies have been reported to induce cytotoxicity and apoptosis in rat mesangial cells (50). It remains to be determined whether their binding to HMC can affect mesangial cell functions, such as the clearance of immune deposits, the production of cytokines and growth factors, or contractile properties that can affect intrarenal hemodynamics, cell proliferation, and apoptosis.

	Acknowledgments

 
This work was supported by the Hong Kong Research Grants Council Earmarked Research Grant (338/041/0023) and the University of Hong Kong CRCG Grant (337/041/0054). We are grateful to Drs. PC Tam and SM Chu and their surgical teams for the collection of renal tissues. Part of this work was presented at the 32^nd Annual Meeting of the American Society of Nephrology, Miami, Florida (J Am Soc Nephrol 10: 508A, 1999).

	Footnotes

 
T.M. Chan and S. Yung contributed equally to this work.

	References

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