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Published ahead of print on September 28, 2005
J Am Soc Nephrol 16: 3281-3294, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2004110917
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Basic Immunology and Pathology

Effect of Human Anti-DNA Antibodies on Proximal Renal Tubular Epithelial Cell Cytokine Expression: Implications on Tubulointerstitial Inflammation in Lupus Nephritis

Susan Yung, Ryan C.W. Tsang, Yuling Sun, Jack K.H. Leung and Tak Mao Chan

Department of Medicine, University of Hong Kong, Queen Mary Hospital, Hong Kong

Address correspondence to: Prof. Tak Mao Chan or Dr. Susan Yang, Department of Medicine, University of Hong Kong, Room 303 New Clinical Building, Queen Mary Hospital, Pokfulam Road, Hong Kong. Phone: 852-2855-4041; Fax: 852-2872-5828; dtmchan{at}hkucc.hku.hk

Received for publication November 17, 2004. Accepted for publication August 9, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study aimed to investigate the effects of human anti-DNA antibodies (Ab) from patients with lupus on renal proximal tubular epithelial cells (PTEC), focusing on alterations in cell morphology and proinflammatory cytokine synthesis. Immunohistochemistry showed increased tubulointerstitial IL-6 expression and IgG deposition in renal biopsies from patients with diffuse proliferative lupus nephritis, not observed in controls or membranous lupus nephritis, which correlated with the severity of inflammatory cell infiltration. Sera from patients with lupus nephritis contained IgG that bound to cultured PTEC. Such binding increased with disease activity and correlated with the level of anti-DNA Ab. Incubation of PTEC with anti-DNA Ab that were isolated during active (active Ab) or inactive (inactive Ab) disease induced IL-6 synthesis, both apically and from the basolateral aspect. This was accompanied by altered cell morphology, increased cell proliferation (P < 0.05), and lactate dehydrogenase release (P < 0.05). The binding of inactive Ab and active Ab to PTEC resulted in differential and sequential upregulation of TNF-{alpha}, IL-1{beta}, and IL-6 secretion (P < 0.05). Early induction of TNF-{alpha} was observed with active Ab; the two then acted synergistically to induce IL-6 secretion. Exposure of PTEC to inactive Ab was associated with modest induction of TNF-{alpha}, which was not involved in downstream induction of other proinflammatory peptides. These data suggest distinct immunopathogenetic mechanisms during disease flare or remission. Conditioned media from human mesangial cells acted synergistically with anti-DNA Ab to induce cytokine secretion in PTEC. Results from these studies underscore the pivotal role of PTEC in the pathogenesis of tubulointerstitial inflammation and fibrosis in lupus nephritis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lupus nephritis (LN) is a severe organ manifestation of systemic lupus erythematosus (SLE) and a major cause of morbidity. Anti-DNA antibodies (Ab) are implicated in the pathogenesis of LN. Their levels correlate with disease activity, and they deposit in the glomerulus and along the tubular basement membrane (1–4). Immune deposition is associated with induction of inflammatory cytokines such as IL-6 (5). IL-6 is synthesized by a variety of cell types that include mesangial cells, epithelial cells, fibroblasts, B cells, and T lymphocytes in response to inflammation, infection, or trauma (6–10) and is involved in the regulation of immune responses, induction of acute-phase protein synthesis, bone metabolism, and hematopoiesis (11–13). In addition, IL-6 can exert either pro- or anti-inflammatory functions (12,14,15). Data from animal experiments have implicated IL-6 in the pathogenesis of SLE (16–18). Disease activity in patients with lupus correlates with IL-6 level in serum, urine, and cerebrospinal fluid, and B lymphocytes from patients with SLE increase the synthesis of anti-DNA antibodies upon stimulation with IL-6 (19–21).

Whereas the immunopathogenesis of glomerular lesions in LN has been studied extensively, less is known regarding the tubulointerstitial lesions, despite the common occurrence of tubulointerstitial disease and their strong association with less favorable long-term renal prognosis (22). Furthermore, it has been suggested that tubulointerstitial inflammation may be less amenable to current immunosuppressive treatment compared with glomerular proliferative changes. Proximal tubular epithelial cells (PTEC) are polarized cells that constitute the predominant cell type within the tubulointerstitium. Although previously considered to be involved mainly in transport of fluid and electrolytes, there is increasing evidence to demonstrate the critical role of PTEC in the immunopathogenesis of various renal parenchymal diseases (23,24), in particular to act as a directional regulator/effector of immune-mediated inflammation and fibrosis (3,23).

The aim of this study was to investigate the pathogenetic mechanisms that pertain to tubulointerstitial inflammation in LN, in particular the effect of anti-DNA Ab on the synthesis and polarized secretion of IL-6 by PTEC at different phases of disease and the factors that mediate the alterations in IL-6 synthesis. Because anti-DNA deposition in the mesangium is a prominent feature in LN, we also examined whether upon stimulation by anti-DNA Ab mesangial cells might elaborate soluble factors that influence IL-6 synthesis by PTEC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Serum Samples
A total of 400 serum samples that were obtained from 210 patients (180 women, 30 men; mean age 45.6 ± 15.4 yr) with biopsy-proven diffuse proliferative LN were screened for Ig binding to PTEC by cellular ELISA (details given below). Sera from 15 patients (11 women and four men; mean age 41.6 ± 8.0 and 43.8 ± 10.5 yr, respectively) that demonstrated high IgG binding to PTEC were selected for anti-DNA Ab isolation and further studies. Each of the selected patients had at least one serum obtained during active disease and another during remission. Active disease was defined by clinical manifestations together with an SLE Disease Activity Index ≥10, and quiescence was confirmed with an SLE Disease Activity Index <4 (25). For investigating the potential effect of immune complexes on PTEC binding activity, in some experiments the serum samples (5 µl diluted in 80 µl of PBS) were incubated with protein G-Sepharose beads (10 µl) for 15 min at room temperature with constant agitation before centrifugation to remove immune complexes before testing by cellular ELISA (26).

Cytochemical and Immunohistochemical Studies on Renal Biopsies
Twelve renal biopsies that showed active diffuse proliferative LN and six renal biopsies that showed pure membranous LN were included. Normal kidney tissue from five patients who underwent nephrectomy for tumor was included as control. Unless otherwise stated, all incubations were for 1 h at 37°C. Paraffin sections were stained with eosin and hematoxylin (Sigma, Tin Hang Technology Ltd, Hong Kong) according to standard procedures. Tubulointerstitial abnormalities including inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis were assessed and scored (0, normal; 0.5, small areas involved; 1, <10% of tubulointerstitium involved; 2, involvement of 10 to 25%; 3, involvement of 25 to 75%; and 4, >75% of tubulointerstitium involved) (27). For detecting IL-6 expression, cryosections (5 µm) were incubated with primary Ab against human IL-6 (1:100 dilution) and incubated with the FITC-conjugated secondary antibody (1:150 dilution) in a darkened humidified chamber (28). For detecting IgG deposition, cryosections were incubated with a biotinylated goat anti-human IgG (dilution 1:50) followed by FITC-conjugated avidin. After washing with PBS, sections were mounted with fluorescence mountant (DAKO, Gene Company, Hong Kong), and epifluorescence was viewed using an Axiovert 135 inverted microscope (Zeiss, Gold Pacific Ltd, Hong Kong). All samples were coded and evaluated without knowledge to the clinical information. Six separate images of the tubulointerstitium were taken for each sample. The extent and the intensity of staining were scored semiquantitatively (1, 0 to <5%; 2, 5 to 25%; 3, 25 to 75%; and 4, >75%), and a mean score was calculated for each sample (29).

Cell Culture
Human Renal PTEC.
Primary cultures of PTEC were obtained from normal renal cortical tissue (30) and maintained in DMEM/Ham’s F12 medium (Invitrogen Life Technologies, Hong Kong) supplemented with 10% FCS. PTEC were characterized by their epithelial, cobblestone morphology and immunohistochemistry (positive for alkaline phosphatase, cytokeratin, and vimentin and negative for von Willebrand factor). Cells were passaged at a split ratio of 1:3, and all experiments were performed on cells of the second passage that had been growth arrested for 72 h. The mechanism of IL-6 induction was subsequently investigated in HK-2 cells (American Type Culture Collection, Manassas, VA), which are normal PTEC immortalized by transduction with the human papilloma virus 16 E6/E7 genes. HK-2 cells of the 10th through 15th passages that had been growth arrested for 72 h were used in experiments. Similarity between normal PTEC and HK-2 cells with regard to cell morphology and functions has been demonstrated previously (31). Our own studies also showed identical results between PTEC and HK-2 cells with regard to Ig binding and IL-6 secretion upon stimulation with sera from patients with LN (data not shown).

Human Mesangial Cells.
Primary cultures of human mesangial cells (HMC) were established from nephrectomized kidneys (26) and maintained in RPMI 1640 medium supplemented with 15% FCS. HMC were characterized by their stellate morphology, ability to form hillocks, and immunohistochemical staining (positive for vimentin and negative for cytokeratin and von Willebrand factor).

Isolation of Human Polyclonal anti-DNA Ab and Measurement of Anti-DNA Activity
Polyclonal anti-DNA Ab were isolated from sera of patients with LN using sequential affinity chromatography as described previously (26). The purity of IgG anti-DNA immunoglobulins was confirmed by 10% SDS-PAGE and anti-DNA assay, and the absence of immune complexes was confirmed with polyethylene glycol assay (26). Anti-DNA activity in serum samples and isolated anti-DNA Ab preparations was determined using a commercial ELISA according to the manufacturer’s instructions (Microplate autoimmune anti-DNA quantitative ELISA; Bio-Rad, Hong Kong). The limits of detection were 20 and 1000 IU/ml, respectively, and samples that gave a value >60 IU/ml were considered positive. Anti-DNA Ab that were isolated during active and inactive disease are referred to as active Ab and inactive Ab, respectively, in this article.

Measurement of IgG Concentration in Serum Samples or Anti-DNA Ab Preparations
This was measured by ELISA as described previously (26). Briefly, goat anti-human IgG (10 µg/ml, 200 µl; Biosource International, Hong Kong) in 0.05 M carbonate buffer (pH 9.5) was coated onto 96-well microtiter plates at 4°C overnight. The plates were washed thrice with PBS/0.1% Tween-20 and blocked with 3% BSA for 1 h at 37°C. After washing with PBS, samples (starting dilution 1:1000) or standards (0 to 500 ng/ml) were added in triplicate in serial dilutions and incubated for 1 h at 37°C. The plates were washed thrice in PBS, and alkaline phosphatase–conjugated goat anti-human IgG (5 µg/ml) was added and incubated for 1 h at 37°C. Bound Ig was detected by the addition of para-nitrophenol phosphate, and the absorbance was read at A405/420. Previous experiments showed <5% cross-reactivity of the Ab with other Ig isomers. Intra-assay and interassay coefficients of variance were 5.4 ± 1.9 and 6.2 ± 2.5%, respectively.

Cellular ELISA to Determine Ig Binding to PTEC
PTEC were seeded into 96-well tissue culture plates at a density of 10,000 cells/cm2 in DMEM/Ham’s F12 that contained 10% FCS until 90% confluent. The cells then were depleted of serum for 72 h before fixing with 1% paraformaldehyde in PBS (pH 7.5) for 5 min at room temperature (26). The cells were washed thrice with PBS between incubations, and incubations were for 1 h at 37°C. PTEC were blocked with BSA and incubated with serum samples (dilution 1:100), polyclonal anti-DNA Ab preparations, or control IgG (10 µg/ml final IgG concentration). This IgG concentration was chosen on the basis of results showing a dose-dependent response up to 12 µg/ml for IgG (Figure 1A). In some experiments, anti-DNA Ab were premixed with DNA at concentrations of 0 to 10 µg/ml for 2 h at 37°C before incubation with PTEC to investigate the effect of DNA on the subsequent binding of Ig to the cells (26). The cells then were incubated with goat anti-human IgG F(ab) conjugated with alkaline phosphatase (5 µg/ml). This was followed by the addition of para-nitrophenol phosphate, and the optical density was determined at A405/420 when the positive control reached an optical density of 1.5. Serum and isolated anti-DNA Ab from a patient with high PTEC-binding activity were used as positive controls. A standard curve was obtained by plotting mean optical density against the amount of PTEC-bound IgG for samples that were obtained from 30 patients who had LN and showed different degrees of PTEC-binding activity. The amount of IgG that bound to PTEC for each sample, expressed as µg PTEC-bound IgG/µg cellular protein, was determined from the difference in IgG concentration in the tested sample before and after incubation with PTEC. This standard curve (correlation coefficient = 0.93; Figure 1B) was subsequently used to determine the amount of IgG bound to PTEC in control and tested samples in this study. Seropositivity for PTEC binding was defined by readings that exceeded mean ± 3 SD of control. For determining total cellular protein, PTEC that were cultured in 96-well plates were lysed with 4 M urea buffer and 20 mM sodium acetate (pH 6.0) that contained 1% Triton X-100 (50 µl). Protein content then was measured using a modified Lowry assay (BioRad, Hong Kong).



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  		Figure 1. Dose-response curve showing the relationship between optical density (OD) indicating IgG bound to proximal tubular epithelial cells (PTEC) and the IgG concentration added to the cells (A) and standard curve relating OD to the cell-bound IgG in the cellular ELISA (B). PTEC were incubated with serum samples at various IgG concentrations (0 to 100 µg/ml), and the amount of IgG bound to the cells was determined by cellular ELISA. Data represent mean OD values of samples ± SD. The standard curve was constructed by plotting OD against IgG bound to PTEC, using samples with established different PTEC-binding activities.

 
Detection of IgG Binding to PTEC by Flow Cytometry
Confluent PTEC were incubated with 0.05% trypsin and 0.02% EDTA for 5 min at 37°C and neutralized with trypsin inhibitor (1 mg/ml) in PBS. PTEC then were cultured in suspension for 4 h, pelleted by centrifugation at 1500 x g for 10 min, washed thrice with PBS, and incubated with anti-DNA Ab preparations or control IgG for 30 min at 4°C in Krebs-Ringer bicarbonate buffer that contained 1% BSA (26). Unbound IgG was removed by washing with the above buffer. PTEC then were 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 that contained 0.5% formaldehyde. IgG binding was analyzed by flow cytometry (Coulter Epics XL Flow Cytometer; Beckman-Coulter Hong Kong Ltd., Hong Kong) with XL System II software, counting 5000 cells for each sample.

Assessment of PTEC Proliferation and Release of Lactate Dehydrogenase
PTEC were seeded into 35-mm dishes at a density of 10,000 cells/cm2 and cultured for 24 h in medium that contained 10% FCS. Cells were washed twice with PBS to remove unattached cells and serum starved for 72 h before incubation with normal human IgG or polyclonal anti-DNA Ab for periods up to 48 h. At select time points, PTEC were trypsinized with 0.05% trypsin/0.02% EDTA and counted using a Neubauer chamber to assess proliferation.

For measurement of lactate dehydrogenase (LDH) release, confluent PTEC in 96-well tissue culture plates were exposed to experiment or control conditions for up to 48 h. Supernatants were collected and centrifuged for 10 min at 2000 x g, and the level of LDH was measured using a commercially available cytotoxicity kit (Boehringer Mannheim, Mannheim, Germany). Results were expressed as the percentage of total intracellular LDH released, the latter determined by lysis of cell monolayer using 2% Triton X-100.

Stimulation of PTEC or HMC with Anti-DNA Ab and Subsequent Cytokine Expression
PTEC were incubated with sera from patients with LN or control sera from healthy subjects (1:100 dilution), isolated polyclonal anti-DNA Ab or normal IgG, in separate experiments (10 µg/ml final IgG concentration) in the presence or absence of DNA (0 to 10 µg/ml) or histones (0 to 10 µg/ml) for periods up to 72 h. After stimulation, the culture supernatant then was decanted, and its level of IL-6, IL-1{beta}, and TNF-{alpha} was measured. For examining the mechanisms of induced IL-6 secretion, cells were incubated with actinomycin D (5 µg/ml) or cycloheximide (5 µg/ml) for 1 h before the addition of the respective "stimulants." Our preliminary experiments demonstrated maximum inhibition of gene transcription and mRNA translation at these doses, without significant cytotoxic effect. In separate experiments, cells were incubated with neutralizing antibodies to IL-6 (200 ng/ml) or TNF-{alpha} (200 ng/ml) or IL-1 receptor antagonist (200 ng/ml) for 1 h before stimulation with anti-DNA Ab preparations or control IgG. For investigating potential additive or synergistic effect of anti-DNA Ab and proinflammatory cytokines on induced IL-6 secretion, cells were incubated with exogenous IL-6 (10 ng/ml), IL-1{beta} (1 ng/ml), or TNF-{alpha} (1 ng/ml) in the presence or absence of control IgG or inactive Ab/active Ab. We also investigated whether upon stimulation with anti-DNA Ab PTEC or HMC might express soluble factors that could influence IL-6 secretion in PTEC. In these experiments, culture supernatants from cells that had been incubated with anti-DNA preparations or normal IgG (10 µg/ml) for 24 h were collected, centrifuged at 2000 x g for 10 min to remove cell debris, then passed through an Ultra-15 centrifugal filter unit with a 100-kD molecular weight cutoff (Millipore Asia, Hong Kong) to remove immunoglobulins. The filtrate then was incubated with PTEC in the absence of anti-DNA Ab, and the subsequent IL-6 secretion from PTEC was examined.

For examining the polarity of IL-6 secretion, PTEC were cultured on six-well transparent polyethylene terephthalate cell culture inserts (0.4 µm pore size; Becton Dickinson, Bio-Gene, Hong Kong) until confluent. Cells were growth arrested for 72 h before apical (1.5 ml final volume) or basal (3 ml final volume) stimulation with anti-DNA Ab, control IgG, or serum-free medium for periods up to 48 h. Supernatants in the upper and lower chambers were collected for IL-6 measurement, and the cells were lysed as described above to determine total cellular protein content.

Measurement of IL-1{beta}, IL-6, and TNF-{alpha} Concentration in Culture Supernatant
After incubation of PTEC with test serum or Ig samples, the supernatant was decanted and centrifuged at 2000 x g for 10 min to remove cell debris, and the level of IL-6, IL-1{beta}, and TNF-{alpha} was measured using respective commercial ELISA kits (Pharmingen, Bio-Gene, Hong Kong). Lower and upper detection limits were 5 and 300 pg/ml, respectively, for IL-6 and IL-1{beta} and 15 and 1000 pg/ml, respectively, for TNF-{alpha}.

Statistical Analyses
All experiments were repeated three times. Results are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego, CA). Active and inactive serum or Ig samples were compared using ANOVA. Correlation between PTEC binding by anti-DNA Ab and IL-6 secretion was examined using the Spearman method. Two-tailed P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IgG Deposition and IL-6 Expression in Renal Biopsies of LN
Significant IgG deposition with a fine granular distribution along the tubular basement membrane was observed in seven of 12 renal biopsies that showed diffuse proliferative LN and one of six renal biopsies that showed pure membranous LN (Figure 2A). Increased IL-6 expression along the tubular basement membrane was demonstrated in 10 (83.3%) of 12 renal biopsies that showed diffuse proliferative LN and two (33.3%) of six biopsies that showed membranous LN. Tubulointerstitial IL-6 staining score correlated with the scores for IgG binding and tubulointerstitial abnormalities and with the PTEC-binding activity, IL-6 level, and anti-DNA Ab level in serum samples (Figure 2B).



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  		Figure 2. Immunohistochemical staining for IgG and IL-6 in renal biopsies showing lupus nephritis (LN; A). Specimens included control renal tissue from patients who underwent nephrectomy for tumor (a, d, and g), diffuse proliferative LN (b, e, and h), and pure membranous LN (c, f, and i). Deposition of IgG in the tubulointerstitium (a through c) and expression of IL-6 (d through f) were investigated, and positive staining is depicted by arrows. Hematoxylin- and eosin-stained sections were also included (g through i). All 12 sections were scored by an observer who was blinded to the clinical data, and correlations among IgG deposition, IL-6 expression, tubulointerstitial abnormalities, serum IL-6 levels, anti-DNA antibody (Ab) titers, and serum PTEC-binding activity are presented (B). Magnification, x400 in A.

 
PTEC-Binding IgG in Serum Samples from Patients with LN
By cellular ELISA with PTEC as substrate, significant IgG binding to PTEC was observed with serum samples from patients with diffuse proliferative LN compared with sera from healthy control subjects, especially during active disease (Figure 3A). Removal of immune complexes from serum samples did not affect the binding of IgG to PTEC (Table 1). Seropositivity rates (as defined by IgG PTEC-binding activity more than mean ± 3 SD of control or 4.3 µg of PTEC-bound IgG/µg cellular protein) were 60.0 and 93.3% in inactive and active LN sera, respectively. Disease activity was associated with higher levels of circulating anti-DNA Ab, total IgG, IgG PTEC-binding activity, and IL-6 levels (Table 2). IgG binding to PTEC also correlated with the levels of circulating anti-DNA Ab (Figure 3B).



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  		Figure 3. Comparison of PTEC-binding activity in serum samples from patients with LN (A). Paired serum samples were obtained during active disease or remission from 15 patients with diffuse proliferative LN. The amount of IgG that bound to PTEC was determined by cellular ELISA. Data are represented as median (horizontal line), 25th and 75th percentile (box), and range (the whiskers extending below and above represent the lowest and highest values for each group; A and D). *P < 0.01, control versus inactive sera; **P < 0.001, control versus active sera and active versus remission sera. Correlation was noted between PTEC-binding activity and anti-DNA Ab titers in serum samples from patients with LN, during remission ({circ}) or active disease (•; B). Comparison of PTEC-binding activity in anti-DNA Ab and whole sera showed enrichment of binding in anti-DNA Ab compared with their corresponding whole sera, during both active disease and remission (C). Horizontal bars represent mean values. Comparison of PTEC-binding in non–anti-DNA Ig and anti-DNA Ab from patients with inactive and active disease showed that PTEC-binding activity was restricted to the anti-DNA fraction (D). Anti-DNA Ab were isolated from paired serum samples that were obtained during active disease or remission from 15 patients with diffuse proliferative LN. The amount of IgG that bound to PTEC in different samples was determined by cellular ELISA.

 

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  		Table 1. Comparison of PTEC-binding IgG in serum samples from patients with LN before and after removal of immune complexesa

 

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  		Table 2. Comparison of PTEC-binding IgG level, total IgG concentration, anti-DNA Ab titer, and IL-6 concentrations in serum samples obtained from patients with LN obtained during active disease or remissiona

 
Binding of Polyclonal Human Anti-DNA Ab to PTEC
We demonstrated previously that anti-DNA Ab accounted for the IgG binding to HMC (26). In this study, polyclonal anti-DNA Ab were isolated from sequential sera of 15 patients who had biopsy-proven diffuse proliferative LN and showed significant IgG PTEC-binding activity in serum. These anti-DNA Ab showed significant binding to PTEC compared with control IgG. Anti-DNA Ab preparations showed enhanced PTEC-binding activity compared with the corresponding original whole sera (Figure 3C). The non–anti-DNA Ig fractions of the corresponding samples did not show significant IgG binding to PTEC (P = 0.156 and 0.159 comparing normal IgG with non–anti-DNA IgG from inactive or active sera, respectively; Figure 3D). In all patients, active Ab showed increased binding to PTEC compared with inactive Ab. Results of cellular ELISA were confirmed with flow cytometry (mean fluorescence intensity for control IgG, inactive Ab, and active Ab were 6.9, 47.0, and 86.2%, respectively). Previous incubation of anti-DNA antibodies with exogenous DNA (10 µg/ml) inhibited their subsequent binding to PTEC (2.8 ± 0.8, 3.1 ± 1.1, and 3.5 ± 1.5 µg PTEC-bound IgG/µg cellular protein for control IgG, inactive Ab preincubated with DNA, and active Ab preincubated with DNA, respectively).

Effect of Anti-DNA Ab on PTEC Morphology, Proliferation, and LDH Release
Incubation of PTEC with active Ab for 24 h induced changes in PTEC phenotype: the cells became elongated to spindle-shaped, with a concomitant reduction in cell–cell contact and cell attachment (Figure 4A, right). Inactive Ab induced similar morphologic changes but to a lesser extent (Figure 4A, middle). Control human IgG (10 µg/ml) had no effect on normal PTEC morphology (Figure 4A, left). The phenotypic changes that were induced by anti-DNA Ab were associated with increased cell proliferation and LDH release (Figure 4, B and C).



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  		Figure 4. Effect of polyclonal anti-DNA Ab from patients with LN on PTEC morphology (A). Cultured PTEC were incubated with control IgG (left) and anti-DNA Ab that were obtained during remission (middle) or active disease (right) for 24 h. The morphology was monitored with phase-contrast microscopy. The normal cobblestone epithelial morphology was preserved in cells that were incubated with control IgG (A). PTEC that were incubated with anti-DNA Ab became elongated (arrow), and some showed reduced cell attachment (*), especially with active Ab. Effect of anti-DNA Ab on PTEC proliferation (B) and lactate dehydrogenase (LDH) release (C). Twenty percent confluent PTEC were incubated with control IgG ({square}) and anti-DNA Ab that were obtained during remission ({square}) or active LN () for up to 48 h. Thereafter, cells were trypsinized and counted using a Neubauer chamber. LDH release from PTEC after incubation with control IgG ({square}) and anti-DNA Ab that were obtained during remission ({square}) or active LN () was measured according to the manufacturer’s instructions and expressed as percentage of total releasable LDH. (C) Cells before exposure to anti-DNA Ab or control IgG or basal secretion of LDH, respectively. Data represents mean ± SD of three experiments. *P < 0.01, {ddagger}P < 0.001, versus control IgG. Magnification, x200 in A.

 
Effect of Anti-DNA Ab on IL-6 Synthesis by PTEC
Incubation of PTEC with anti-DNA antibodies from patients with LN induced IL-6 secretion, which was not observed when cells were incubated with the non–anti-DNA Ig fractions from corresponding serum samples (Figure 5A). Active Ab induced more IL-6 compared with inactive Ab in 14 of the 15 patients. Data from PTEC that were cultured on inserts for up to 48 h showed that when exposed to normal IgG, there was slight time-dependent IL-6 secretion, predominantly from the basolateral aspect (Figure 5B). Apical or basolateral stimulation with anti-DNA Ab resulted in increased IL-6 secretion mostly into the same compartment as the stimulant, and more profound induction was observed with active Ab compared with inactive Ab (Figure 5B).



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  		Figure 5. Effect of anti-DNA Ab and non–anti-DNA Ig on IL-6 secretion in PTEC, during active disease or remission (A). Confluent growth-arrested PTEC were stimulated with control IgG, isolated anti-DNA Ab, or non–anti-DNA Ig for 24 h. The supernatant was decanted and assayed for total IL-6 secretion using a commercial ELISA. Horizontal bars represent mean values. In separate experiments, directional secretion of IL-6 was investigated in cells that were cultured on inserts and stimulated either apically (left) or on their basolateral aspect (right) with control IgG ({square}), inactive Ab ({cjs2110}), or active Ab ({square}) for periods up to 48 h. *P < 0.05, **P < 0.001, versus control IgG. Anti-DNA Ab preparations showed enrichment in the ability to induce IL-6 secretion compared with their corresponding whole sera (C). Horizontal bars represent mean values.

 
When tested at the same IgG concentration, purified anti-DNA Ab induced more IL-6 secretion compared with the corresponding original whole serum (Figure 5C). Induction of IL-6 secretion correlated with PTEC-binding activity and anti-DNA Ab levels of the serum samples (Figure 6). Induction of IL-6 by anti-DNA Ab was dependent on both de novo IL-6 gene transcription and translation (Figure 7). Altering the concentration of DNA or histone that was added concomitantly with anti-DNA Ab had no effect on IL-6 induction (data not shown).



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  		Figure 6. Correlation between induction of IL-6 secretion and PTEC-binding activity (A) or anti-DNA Ab titer (B) in serum samples from patients with LN during remission ({circ}) and during active disease (•).

 


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  		Figure 7. Effect of actinomycin D or cycloheximide on the induction of IL-6 secretion in PTEC by anti-DNA Ab. Confluent growth-arrested PTEC were incubated with actinomycin D (5 µg/ml) or cycloheximide (5 µg/ml) for 1 h before stimulation of cells with anti-DNA Ab for another 24 h. The results show that IL-6 induction by anti-DNA Ab is dependent on de novo gene transcription and translation. Data represent mean values and SD of three individual experiments. *P < 0.01, with or without drug treatment, or compared with control IgG.

 
Mechanisms of PTEC IL-6 Induction by Anti-DNA Ab
Induction of IL-6 by inactive or active Ab was preceded by induction of TNF-{alpha} and followed by induction of IL-1{beta} in PTEC (Figure 8). Because previous studies have demonstrated that IL-1{beta} and TNF-{alpha} can induce IL-6 secretion in mesangial or mesothelial cells (32,33), we investigated whether they mediated the induction of IL-6 in PTEC stimulated with anti-DNA Ab. Whereas induction of IL-6 by inactive Ab was not altered by TNF-{alpha} neutralizing Ab (Figure 9), there was a 37.1 ± 2.4% reduction with IL-1 receptor antagonist. Previous incubation of PTEC with IL-6 neutralizing antibody also abrogated the subsequent induction of IL-6 by inactive Ab (P < 0.001). In contrast, induction of IL-6 by active Ab was reduced with TNF-{alpha} neutralizing antibody, IL-1 receptor antagonist, or IL-6 neutralizing antibody.



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  		Figure 8. Effect of anti-DNA Ab on IL-1{beta} and TNF-{alpha} secretion in PTEC. Confluent growth-arrested PTEC were stimulated with control IgG ({diamondsuit}), or anti-DNA Ab that were obtained during remission ({circ}) or active LN (•) for up to 48 h. At select time points, the culture medium was decanted and centrifuged to remove cell debris, and the levels of IL-1{beta} (A) and TNF-{alpha} (B) were measured using a commercial ELISA. Data represent mean ± SD of three separate experiments. *P < 0.01, versus control at the same time point.

 


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  		Figure 9. Role of IL-6, IL-1{beta}, or TNF-{alpha} in mediating the induction of IL-6 in PTEC by anti-DNA Ab. PTEC were incubated with serum-free medium (SFM; {square}), control IgG ({square}), anti-DNA Ab that were obtained during remission () or active LN ({blacksquare}), in the presence or absence of neutralizing Ab to IL-6 or TNF-{alpha}, or IL-1 receptor antagonist. In some experiments, exogenous IL-6, IL-1{beta}, or TNF-{alpha} was added together with anti-DNA Ab to investigate potential synergistic or additive effects on the induction of IL-6 secretion. *P < 0.05 versus control IgG. Data represent mean ± SD of three individual experiments.

 
For investigating potential additive or synergistic effect of TNF-{alpha}, IL-1{beta}, or IL-6 on IL-6 induction by anti-DNA Ab, exogenous cytokines were added to PTEC in the presence or absence of anti-DNA Ab. All three cytokines induced IL-6 secretion in PTEC, and IL-1{beta} had the most prominent stimulatory effect. Whereas both active Ab and inactive Ab interacted synergistically with IL-1{beta} and with IL-6 in stimulating PTEC IL-6 synthesis there was only a synergistic effect between TNF-{alpha} and active Ab.

Effect of Anti-DNA Ab-Stimulated Mesangial Cell/PTEC Culture Supernatant on PTEC IL-6 Secretion
In the absence of anti-DNA Ab ("basal" condition), PTEC and HMC secreted similar concentrations of IL-6 into the supernatant. Active Ab induced more IL-6 secretion from PTEC (64.3-fold increase) compared with HMC (22.5-fold increase; Figure 10). Conditioned supernatant from anti-DNA Ab-stimulated PTEC or HMC induced IL-6 secretion in PTEC by 31.4- and 23.4-fold, respectively, compared with conditioned supernatant obtained from control IgG-stimulated PTEC or HMC. Such induction was mediated through TNF-{alpha}, IL-1{beta}, and especially IL-6, as demonstrated by the observed reduction in IL-6 secretion in the presence of TNF-{alpha} neutralizing antibody, IL-1 receptor antagonist, and IL-6 neutralizing antibody. Similar results were obtained when conditioned supernatant from anti-DNA Ab–stimulated PTEC or HMC was added to HMC.



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  		Figure 10. Effect of conditioned culture supernatants from human mesangial cells (HMC) or PTEC after stimulation with anti-DNA Ab on the subsequent induction of PTEC IL-6 by anti-DNA Ab. Confluent growth-arrested PTEC and HMC were incubated with serum-free DMEM/Hams F-12 medium (SFM) in the presence or absence of control IgG or anti-DNA Ab that were derived from 10 patients with active LN (10 µg IgG/ml) for 24 h. The conditioned culture supernatant then was retrieved, centrifuged at 2000 x g for 10 min to remove cell debris, and passed through an Ultra-15 centrifugal filter unit with a 100-kD molecular cutoff to remove remaining IgG or anti-DNA Ab. The IgG level in the filtrate was measured to confirm that it was negligible. Fresh PTEC or HMC then were incubated with this conditioned medium (CM) for 24 h, and the induction of IL-6 was assessed. In some experiments, neutralizing Ab to IL-6 (200 ng/ml) or TNF-{alpha} (200 ng/ml) or IL-1 receptor antagonist (200 ng/ml) was added to the cells for 1 h before the addition of conditioned culture supernatant to determine IL-6 induction. Data represent mean ± SD of three individual experiments. {ddagger}P < 0.05 and *P < 0.001, versus cells that were exposed to control IgG.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
High levels of circulating anti-DNA Ab, mesangial cell proliferation, Ig deposition within the glomerulus, and local inflammation are characteristic features in severe LN (1–4). Whereas the glomerular abnormalities have attracted much attention, the immunopathogenetic mechanisms that lead to tubulointerstitial inflammation and damage in LN remain obscure (3). This is ironic because tubulointerstitial damage is an important poor prognostic indicator for long-term renal function (22), and it may be less amenable to treatment compared with glomerular lesions. Tubulointerstitial involvement, characterized by inflammatory cell infiltration and induction of cytokine expression and followed by tubular atrophy and interstitial fibrosis, increases with the severity of LN. PTEC constitute the predominant cells within the tubulointerstitium and play a pivotal role in the initiation of the renal inflammatory response. Once considered to play only a physiologic role within the kidney, there is now compelling evidence to underscore the importance of PTEC in renal immunological functions such as processing and presentation of foreign antigen and synthesis of proinflammatory cytokines (34–36). Furthermore, studies have shown that the induced synthesis of IL-6 by PTEC during renal disease contributes to tubular inflammation and progression of tubulointerstitial nephritis (35). Other investigators have reported that anti-DNA Ab could upregulate IL-6 gene expression in mesangial cells (37). In this study, we investigated the effect of human polyclonal anti-DNA Ab that were isolated from patients with LN on IL-6 synthesis by PTEC.

The clinical relevance of these studies is highlighted by the demonstration of tubulointerstitial IgG deposition and IL-6 expression in renal biopsies of LN, in particular with diffuse proliferative disease. Furthermore, although the concordance was not absolute, an overall correlation between the magnitude of the two was indicated by the immunohistochemical scores. The pathogenetic importance of IL-6 was evident from the finding that IL-6 expression correlated with inflammatory cell infiltration. Increased IL-6 expression has been demonstrated in resident and infiltrating cells in various renal diseases such as LN, diabetic nephropathy, and IgA nephropathy (36,38,39). It is noteworthy that increased tubulointerstitial IL-6 in LN was attributed primarily to increased IL-6 expression by PTEC, thereby underscoring the pivotal role of PTEC in immunopathogenesis.

To facilitate comparison of Ig binding to PTEC between serum samples or anti-DNA Ab preparations, we expressed the results of the cellular ELISA as the quantity of PTEC-bound IgG normalized to the quantity of cellular protein. Our results showed that serum samples from patients with LN contained IgG that could bind to PTEC. We previously reported that such cellular binding by lupus IgG occurred with mesangial cells and PTEC but not significantly with other cell types (26). The correlation among PTEC-binding activity, disease activity, and the level of circulating anti-DNA Ab suggested that the last could contribute to the PTEC-binding Ig. The mechanisms by which anti-DNA Ab bind to mesangial cells, endothelial cells, mesothelial cells, and mononuclear cells have been investigated (9,26,40,41), but the actions of anti-DNA Ab on PTEC remain undefined. We have observed that incubation of PTEC with anti-DNA Ab resulted in altered cell morphology, increased cell proliferation, and LDH release. These effects were more pronounced with anti-DNA Ab that were obtained during active disease. That both cell proliferation and LDH release were increased upon biologic insult is intriguing but has been reported previously in PTEC. Burton et al. (42) reported such findings when cultured renal tubular cells were stimulated with serum proteins from patients with proteinuric diseases and suggested that these reflected cellular injury and the subsequent recovery process. Data from animal studies have shown diminished apoptosis in SLE (43,44) and associated abnormal cell proliferation within the glomerulus (44). Whether the increased PTEC proliferation induced by anti-DNA Ab is related to diminished apoptosis remains to be investigated.

IL-6 is a pleiotropic cytokine produced by a wide variety of cells in response to trauma, infection, and inflammation. IL-6 promotes inflammation through its effects on B and T lymphocyte activation and differentiation, as well as the induction of acute-phase reactants (11,45). An anti-inflammatory role of IL-6 has also been suggested (15). The pathogenetic importance of IL-6 in LN is underscored by its stimulatory actions on B lymphocytes, thereby increasing the level of anti-DNA Ab (16,17,45). Increased serum IL-6 levels correlate with lupus activity (19,20), and LN is associated with increased glomerular IL-6 expression (36). Interruption of IL-6 signaling inhibits the onset of autoimmune kidney disease in (NZB x NZW)F1 mice (46). Furthermore, recent studies have shown that IL-6 can activate the transcription of the IFN-inducible gene Ifi202, which is a major genetic contributor to the pathogenesis of SLE (47). Results from our studies showed that anti-DNA Ab from patients with LN induced IL-6 synthesis in PTEC, and such induction was dependent on de novo synthesis of IL-6 transcript and protein synthesis. Because we compared anti-DNA Ab that were isolated during active disease or remission at identical IgG concentration, their difference in the ability to induce IL-6 suggests qualitative variations of Ab functions that correlate with clinical activity. This hypothesis is in keeping with the different degrees of binding to PTEC between active Ab and inactive Ab. It is of interest to note that the increase in IL-6 secretion upon stimulation with anti-DNA Ab was directional and occurred in the same compartment as the stimulant. Consequently, exposure of the basolateral aspect of PTEC to anti-DNA Ab induced predominantly basolateral IL-6 secretion, the in vivo analogy of which suggests that there is increased IL-6 in the interstitial space, thereby promoting local inflammatory processes including the recruitment of inflammatory cells. Whether the cytokines that are secreted from the luminal aspect of PTEC could be reabsorbed and contribute to tubulointerstitial abnormalities remains to be determined.

Induction of PTEC IL-6 secretion by anti-DNA Ab was accompanied by increased secretion of IL-1{beta} and TNF-{alpha}. It is of interest that active Ab and inactive Ab show distinct mechanisms in IL-6 induction. Both IL-1{beta} and IL-6 are involved in IL-6 induction by active or inactive Ab, but TNF-{alpha} plays a role only during active disease. In addition, these cytokines act synergistically with anti-DNA Ab in amplifying the inflammatory response. This synergism is again absent between inactive Ab and TNF-{alpha}. These observations provide insight into the pathogenetic mechanisms and the interaction between different cytokines at different phases of disease. They also highlight the heterogeneity of anti-DNA Ab within the same patient between disease flare and remission. Other investigators have reported on the induction of IL-6 in rat mesangial cells by anti-DNA Ab, although, unlike our results, they did not observe any increase in TNF-{alpha} expression (37). This discrepancy can be attributed to differences in species and experimental protocols. Clinical and animal studies have highlighted the exaggerated renal synthesis of TNF-{alpha} in LN (36,48–50). TNF-{alpha} can exert diverse effects that vary according to the immunologic milieu at different stages of disease (51). Increased TNF-{alpha} expression has been associated with active LN (36,48–50,52), whereas other studies have shown TNF-{alpha} to possess immunosuppressive properties (53). Our results show that the induction of TNF-{alpha} by anti-DNA Ab occurred earlier than other cytokines. In addition, the kinetics of TNF-{alpha} induction seem to be different between active and inactive Ab. Together with data that showed the ability of TNF-{alpha} to induce IL-1{beta} in various cell types, the induction of TNF-{alpha} in PTEC by anti-DNA Ab could represent an early crucial step that leads to downstream events that ultimately culminate in inflammatory damage of the tubulointerstitium.

Pathogenetic mechanisms within different portions of the renal parenchyma may not occur in isolation. Glomerular and tubulointerstitial abnormalities both can be prominent in LN, especially with diffuse proliferative LN. We therefore investigated for potential interaction between mesangial cells and PTEC under the influence of anti-DNA Ab. Our results show that upon stimulation with anti-DNA Ab, both PTEC and HMC secrete soluble factors into the supernatant, which in turn stimulates cytokine synthesis in the other cell type. Moreover, when tested at identical anti-DNA IgG concentrations, PTEC show a more prominent IL-6 induction compared with mesangial cells. These findings demonstrate the importance of PTEC in the immunopathogenesis of LN, in terms of both its magnitude of inflammatory responses and its being a downstream effector under the influence of other resident kidney cells. Our data also suggest a bidirectional communication between the glomerulus (mesangial cells) and the tubulointerstitium via PTEC, with IL-6 as an important mediator. Serum samples from patients with active lupus contain multiple cytokines. However, it is unlikely that the observed induction of cytokine synthesis in PTEC and HMC by anti-DNA Ab preparations in our experiments was due to the presence of cytokines in the Ab preparations, because their isolation involved affinity purification and elution with high salt concentration. In addition, no significant level of IL-6, IL-1{beta}, or TNF-{alpha} could be detected by ELISA.

In conclusion, we demonstrated that anti-DNA Ab in patients with diffuse proliferative LN can bind to PTEC and stimulate the synthesis of proinflammatory cytokines IL-6, IL-1{beta}, and TNF-{alpha}. These changes in cell function induced by anti-DNA Ab are accompanied by altered morphology, proliferation, and viability. The distinct pathways of cytokine induction during active disease and remission probably relate to the heterogeneity in Ab populations. The ability of inactive Ab to induce cytokine induction in PTEC is intriguing, because it suggests ongoing inflammation within the tubulointerstitium despite clinical quiescence. Although the level of inflammation is significantly lower than that observed during active flare, it may account, in part, for the requirement of maintenance therapy in patients with lupus during inactive disease. The synergism among different cytokines, anti-DNA Ab, and different resident kidney cells suggests an interrelated network in mediating inflammatory damage to the tubulointerstitial compartment.


   Acknowledgments
 
Part of this work was presented at the 35th Annual Meeting of the American Society of Nephrology, October 30 to November 4, 2002, in Philadelphia, PA. (J Am Soc Nephrol 2002; 13: 172A).

This work was supported by the Hong Kong Research Grants Council Earmarked Research Grant (HKU7167/99M), CRCG Grant (10205893), and the Wai Hung Charity Foundation. We are grateful to Drs. P.C. Tam and S.M. Chu and their surgical team for the collection of renal tissues and to Dr. K.W. Chan from the Department of Pathology for retrieving renal biopsy specimens.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication November 17, 2004. Accepted for publication August 9, 2005.




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