Skip Navigation


Rheumatology Advance Access originally published online on June 14, 2007
Rheumatology 2007 46(8):1277-1284; doi:10.1093/rheumatology/kem136
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/8/1277    most recent
kem136v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Shui, H.-A.
Right arrow Articles by Chen, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shui, H.-A.
Right arrow Articles by Chen, A.
Related Collections
Right arrow Systemic Lupus Erythematosus and Autoimmunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

LPS-evoked IL-18 expression in mesangial cells plays a role in accelerating lupus nephritis

H.-A. Shui1, S.-M. Ka2, W.-M. Wu4, Y.-F. Lin3, Y.-C. Hou2, L.-C. Su2 and A. Chen2

1Graduate Institute of Medical Sciences, 2Department of Pathology and 3Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center and 4Department of Nutrition and Food Sciences, Fu-Jen University, Taipei, Taiwan, R.O.C.

Correspondence to: A. Chen, Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Gung Road, Taipei, Taiwan, ROC. E-mail: doc31717{at}ndmctsgh.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Statistics
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Systemic lupus erythematosus is occasionally accompanied with bacterial infection. Lipopolysaccharide (LPS) from bacteria can accelerate and exacerbate lupus nephritis (LN) in animal models, but some mechanisms underlying the LPS-induced acceleration are still unclear. First, it is not known whether LPS can stimulate mesangial cells (MCs) to secrete the pro-inflammatory cytokine, interleukin (IL)-18. Second, it is also unclear whether LPS and/or IL-18 can induce MC apoptosis. Here, we attempted to clarify the cause-and-effect relationships between LPS stimulation, IL-18 production and MC apoptosis to address the above questions.

Methods. LPS was used to induce accelerated LN in LN-prone mice. LPS and IL-18 were also used to treat cultured MCs isolated from the mice. IL-18 expression and MC apoptosis were investigated by in situ hybridization, the TUNEL method, reverse transcription– polymerase chain reaction (RT–PCR), western blotting, DNA electrophoresis and flow cytometry. NF{kappa}B was detected by immunofluorescent staining.

Results. In the LPS-accelerated LN mice, we observed co-existence of IL-18 expression, hyperplasia, apoptosis, and activated apoptotic signal transduction in MCs, as well as marked neutrophil infiltration in the glomerulus, especially around the mesangial region. In cultured MCs, LPS greatly enhanced IL-18 expression, but did not induce apoptosis, while mouse IL-18 did not induce apoptosis or activate apoptotic signal transduction in MCs.

Conclusions. We conclude that LPS can evoke IL-18 production in MCs, but neither LPS nor IL-18 directly induces apoptosis or activates apoptotic signal transduction in the cells. We infer that LPS-induced IL-18 production by MCs could be a mediator by which LPS accelerates and exacerbates LN.

KEY WORDS: LPS, IL-18, Lupus nephritis, Mesangial cells, Apoptosis, Accelerated LN


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Statistics
 Results
 Discussion
 Acknowledgements
 References
 
Lupus nephritis (LN) is a frequent organ manifestation of SLE, a disease characterized by the occurrence of many different autoantibodies. LN is also the main cause of morbidity and mortality in SLE. The progression to LN in SLE is very complex [1] and the outcome of LN in SLE is highly variable, ranging from clinically silent nephritis to rapidly progressive crescentic glomerulonephritis with acute renal failure. Many complication factors, including clinical, serological, and histopathological factors, are responsible for the ultimate severity of LN [2].

SLE is occasionally accompanied with bacterial infection [3–6], but the clinical outcomes of bacterial infection in SLE are hard to evaluate due to the interference of medication on patients. Endotoxin, such as lipopolysaccharide (LPS) from Gram-negative bacteria, is a complicating factor which can exacerbate and accelerate LN in mouse models [7]. Both systemic and local effects of LPS are responsible for the LPS-exacerbated LN. The systemic effects of LPS are to induce B cell activation, enhance immune complex deposition [7–9] and increase the expression of ICAM-1, which recruits leucocytes to the kidney [10], while one local effect of LPS on glomeruli is to stimulate mesangial cells (MCs) to produce cytokines, which modulate glomerular inflammation and renal cell survival [11, 12]. Interleukin (IL)-18, a relatively recently discovered cytokine of the IL-1 superfamily, has been shown to play pathogenic roles in LN [13]. So far, it is still unclear whether LPS can stimulate MCs to produce IL-18. If it can, this could provide an insight into the mechanism underlying LPS-accelerated LN.

Besides serving as pro-inflammatory mediators, both LPS and IL-18 are apoptosis-inducing factors that affect the survival of many cells. LPS can directly cause apoptosis of alveolar epithelial, endothelial, neuronal and renal tubular cells [14–17], and IL-18 can directly induce apoptosis of cardiac endothelial and renal tubular cells [18–20]. However, it is still uncertain whether LPS and/or IL-18 has an apoptotic effect on MCs and addressing this question is crucial in demonstrating or excluding a mechanism in which LPS accelerates LN by inducing MC apoptosis, because apoptosis could cause MC loss which compromises histological structure of glomeruli. However, moderate apoptosis is beneficial in LN, since apoptosis is an important mechanism for ameliorating MC hyperplasia in LN [21–23].

In order to evaluate potential local mechanisms in LN involving stimulation by LPS, expression of, and stimulation by, IL-18, and MC apoptosis, in vivo and in vitro studies were carried out in an LPS-induced accelerated-LN mouse model and in cultured MCs from the LN-prone mice. The present study clearly demonstrated that LPS-stimulated MCs produce IL-18 and that this correlates well with severe glomerular lesions, but neither LPS nor IL-18 directly induces MC apoptosis, although they might have indirect apoptotic effects on MCs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Statistics
 Results
 Discussion
 Acknowledgements
 References
 
LPS-induced accelerated-LN mouse model
An LPS-induced accelerated-LN mouse model was established in an 8-week-old female New Zealand black/white (NZB/W) f1 mice by twice weekly intraperitoneal injection of LPS (Sigma, St. Louis, MO, USA) for 6 weeks as described previously [7], then blood and urine samples were collected and the mice killed (n = 7 for each day) and kidney samples were taken for the following studies. All experimental measurements were performed in duplicate. All animal experiments were performed with the ethical approval of the Institutional Animal Care and Use Committee of The National Defense Medical Center, Taiwan and performed according to the ethical rules in NIH Guide for the Care and Use of Laboratory Animals.

Renal function, urine protein and serum autoantibodies
Urine was collected in metabolic cages and blood was collected from the retro-orbital plexus. Serum creatinine was measured by colorimetry using a creatinine detection kit (Sigma 555-A) and blood urea nitrogen (BUN) was measured using a urease assay kit (Sigma 640-A) as described previously [24]. Proteinuria was evaluated by measuring total urine protein using a Pierce BCA protein assay kit (Pierce, IL, USA). Serum autoantibody levels measured using mouse anti-dsDNA and anti-nuclear antibody ELISA kits (Alpha Diagnostic Intl. Inc.) and an ELISA plate reader (Bio-Tek, MA, USA).

Immunofluorescence, in situ hybridization, histopathological evaluation and TUNEL analysis
Renal tissues were snap-frozen or fixed in 10% buffered formalin, then cut into sections for detection of deposited immune complexes by immunofluorescence (IF) and for in situ hybridization (ISH), histopathology evaluation and TUNEL analysis. For IF, frozen sections were air-dried, fixed in acetone for 10 min at room temperature and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or C3 antibodies (Cappel; Organon Teknika, Durham, NC, USA) as described previously [24]. For ISH, RNA probes were generated by in vitro transcription (AmpliScribe T7 and SP6 high yield transcription kits, Epicentre, WI, USA). Briefly, mouse IL-18 cDNA was generated from kidney mRNA by the reverse transcription (RT)–polymerase chain reaction (PCR) (IL-18 primers: forward 5'-ACTGTACAACCGGAGTAATACGG-3' and reverse 5'-TCCATCTTGTTGTGTCCTGG-3'). The product from the PCR reaction was inserted into the pGEM-T vector (Promega, WI, USA) and digoxigenin-labelled RNA probes synthesized using T7 RNA polymerase (Roche, IN, Germany). After hybridization with the probes, alkaline phosphatase-conjugated anti-digoxigenin antibody was used to detect the hybridized probe by colour development in NBT/BCIP solution (Roche). For histopathology, formalin-fixed tissues were stained with hematoxylin and eosin (HE). For TUNEL staining, formalin-fixed sections were stained with an ApopTag Plus Peroxidase in Situ Apoptosis Detection kit (Chemicon) according to the manufacturer's instruction.

Isolation of glomeruli
The glomeruli were extracted from the kidneys using a sieving technique as described previously [25, 26]. Briefly, the kidneys were removed, washed with sterile phosphate-buffered saline (PBS) and decapsulated and the cortex was separated from the medulla. A sample of cortex was cut into 1–2 mm3 blocks, which were ground up in PBS, the suspension passed sequentially through 60, 100 and 200 mesh nets and the glomeruli collected from the surface of the 200 mesh net. The glomerular samples were subjected to total RNA extraction using Trizol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer's instruction for RT-PCR studies and to lysis using RIPA solution [26] for western blotting studies.

Primary cultures of MCs
Female NZB/W mice, 6- to 8-months-old, were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and used as the MC sources. The establishment of primary cultures of MCs from the NZB/W mice was modified from a previously described protocol [27]. Briefly, glomeruli were purified from minced renal cortex by serial sieving through meshes of different pore-sizes, then the glomeruli suspension was digested for 20 min at 37°C with type IV collagenase and the dissociated glomerular cells cultured in RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin and HEPES (10 mM) (GIBCO, Invitrogen, CA, USA). Epithelial cells initially grow faster than MCs, but die after 2–3 weeks and almost pure MCs are then obtained. The cells were plated onto dishes with glass slides and allowed to grow for 16 h in RPMI 1640 medium containing 20% FBS, then for 2 h in RPMI 1640 medium containing 2% FBS. LPS or recombinant mouse IL-18 (MBL, Nagoya, Japan) in RPMI 1640 medium containing 2% FBS was then added for 0, 6, 12 or 24 h and the cells harvested.

Reverse transcriptase (RT)-PCR analysis
Tissue RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). For first-strand cDNA synthesis, 3 µg of total RNA was used in a single-round RT reaction in a final volume of 25 µl of a solution containing 0.9 µl of 50 µM oligo(dT)12–18', 1.0 mM dNTPs, 1x first-strand buffer, 0.4 mM dithiothreitol, 80 U of RNaseout recombinant RNase inhibitor and 300 U superscript II RNase H (Invitrogen, Carlsbad, CA,USA). PCR was performed in a total volume of 15 µl containing 0.9 µl of the cDNA template-containing solution from the RT reaction above, 0.4 µM mouse IL-18 primers (forward: 5'-ACTGTACAACCGGAGTAATACGG-3' and reverse: 5'-TCCATCTTGTTGTGTCCTGG-3') or GAPDH primers (forward: 5'- TCCGCCCCTTCTGCCGATG-3' and reverse: 5'-ACGGAAGGCCATGCCAGTGA -3'), 1x PCR buffer, 0.25 mM dNTPs and 1.5 U of KlenTaq DNA polymerase (Ab Peptides Inc. St. Louis, MO, USA). Amplification was carried out at 94°C for 2 min, then for 25 cycles of 94°C for 45 s, 55°C for 1 min and 72°C for 45 s, and, finally, at 72°C for 10 min. RT-PCR products were resolved on a 1.5% agarose gel containing 0.1% ethidium bromide and 0.5 x TBE buffer, visualized using a gel documentation system (Bio-Rad, Hercules, CA, USA) and analysed by densitometry.

Western blotting
MCs were lysed using RIPA lysis solution and 10 µl of the lysed sample mixed with an equal volume of 2xsample buffer and run on a 10% SDS-PAGE gel; western blotting was then performed as described previously. The primary antibodies used were rabbit PARP antibodies (1/500, Neomarkers Lab Vision Corp.), goat anti-IL-18 (1/100) or ß-actin (1/1000) antibodies, rabbit anti-caspase 3 p20 (1/400), BID (1/400) or Bax (1/400) antibodies, or mouse anti-caspase 8 p20 subunit (1/400) or Bcl-2 (1/400) antibodies (all from Santa Cruz). The secondary antibodies used were HRP-conjugated rabbit anti-goat IgG (1/10 000, Santa Cruz), goat anti-rabbit IgG antibodies (1/10 000, Pierce Il, USA) or rat anti-mouse IgG antibodies (1/1000, Santa Cruz), as appropriate.

DNA fragmentation electrophoresis
Cells (105) were treated with LPS for 0, 6, 12, or 24 h or with 10 mM H2O2 for 6 h as an apoptosis positive control [28]. Low molecular weight DNA was then extracted using a G-DEX Genomic DNA Extraction kit (iNtRON Biotechnology) according to the manufacturer's protocol. A mixture of 13.5 µl of the extracted DNA and 1.5 µl of DNA loading buffer (0.5% bromophenol blue, 50% glycerol, 0.5 mM EDTA) was run on an 1% agarose gel containing 0.1% ethidium bromide and 0.5 x TBE buffer (0.089 M Tris, 0.002 M EDTA, 0.089 M boric acid) and gel images were obtained using a Photo-Documentation System (Viber Lourmat, France).

Assessment of apoptosis by flow cytometry
The cells were collected by centrifugation and washed twice with PBS, then an Annexin V-FITC Apoptosis Detection kit (BD Pharmingen) was used to assess apoptosis according to the manufacturer's instructions. Annexin V-positively and negatively labelled cells were counted on a flow cytometer (FACScalibur) equipped with an argon laser (BD Biosciences) operating at an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The resulting histograms were analysed using an analytical software program (WinMDI).

Immunofluorescence studies of NF-{kappa}B nuclear translocation
MCs were grown on glass slides and fixed with 2% paraformaldehyde for 15 min. For IF staining of NF-{kappa}B p65, the sections were incubated overnight at 4°C with rabbit anti-NF-{kappa}B p65 antibody (Cell Signalling, MA, USA), then for 2 h at room temperature with FITC-conjugated goat anti-rabbit IgG antibody (Cappel; Organon Teknika, Durham, NC, USA). The percentage of positive MCs for nuclear NF-{kappa}B p65 was determined by counting at least 500 cells in each well under a microscope (x400) [29].


   Statistics
Top
 Abstract
 Introduction
 Materials and methods
 Statistics
Results
 Discussion
 Acknowledgements
 References
 
The data are presented as the mean ± SEM. Student's t-test was used for the statistical analysis. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Statistics
 Results
Discussion
 Acknowledgements
 References
 
Clinical manifestations and autoantibody levels
BUN and creatinine levels were significantly higher in the LPS-treated mice than the control mice (228.61 ± 26.79 vs 32.27 ± 4.76 mg/dl and 0.73 ± 0.06 vs 0.35 ± 0.04 mg/dl, respectively; both P < 0.05) (Fig. 1A and B), indicating that LPS accelerated the deterioration of renal clearance function. In parallel, heavier proteinuria was seen in the LPS-treated mice compared with the controls (43.77 ± 1.71 vs 21.84 ± 0.86 mg/ml; P < 0.05) (Fig. 1C), revealing accelerated disruption of the renal filtration barrier by LPS.


Figure 1
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 1. Comparisons of the clinical and immune profiles in control and LPS-treated mice. Levels of (A) BUN, (B) serum creatinine, (C) urinary protein (D) anti-nuclear antibody and (E) anti-dsDNA antibody. The white and black bars indicate the control and LPS groups, respectively. The data are shown as the mean ± SEM and the asterisks indicate a significant difference between the groups (n = 7, *P < 0.05). (F) Typical IF glomerular sections showing deposition of IgG and complement in glomeruli from control and LPS-treated mice.

 
Anti-nuclear antibody titres in the LPS-treated mice were significantly higher than in controls (OD 1.08 ± 0.04 vs 0.54 ± 0.26; P < 0.05) (Fig. 1D), as were anti-ds DNA antibody titres (OD 1.64 ± 0.14 vs 0.384 ± 0.04; P < 0.05) (Fig. 1E). In contrast to the control non-LPS-injected mice, diffuse IgG and C3 deposits were seen in the glomeruli of the LPS-treated mice by IF (Fig. 1F). The higher levels of auto-antibodies in both the serum and glomeruli in the LPS-treated mice show that LPS enhances the autoimmune response [8, 9].

LPS-treated mice show co-existence of IL-18 upregulation, hyperplasia and apoptosis in MCs
Tissue sections stained by ISH, HE and the TUNEL method are shown in Figure 2A. ISH showed that IL-18 mRNA levels were higher in the mesangial area of LPS-treated mice than in controls, indicating that MCs respond to LPS by IL-18 overexpression. HE staining revealed that, in contrast to the histological profile of the control group, glomeruli in the LPS-treated mice showed marked proliferation and morphologically typical apoptosis (apoptotic bodies indicated by thick arrows) in the mesangial area. To confirm the apoptosis in the glomeruli, TUNEL analysis was performed and showed that TUNEL (+) cells were clearly more abundant in the LPS-treated mice than in control mice.


Figure 2
View larger version (70K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 2. Correlation between IL-18 expression and apoptosis in MCs in glomeruli from LPS-treated and control mice. (A) Typical glomerular sections stained by ISH, HE and TUNEL showing IL-18 expression, apoptotic bodies (thick arrows), hyperplasia and DNA fragmentation in MCs, accompanied with neutrophils infiltration (thin arrows). Original magnification, x400. (B) IL-18 mRNA levels in isolated glomeruli from control and LPS-treated mice. Typical gels showing IL-18 and GAPDH mRNA bands are shown in the upper panel, while quantitative IL-18/GAPDH ratios are shown in the lower panel (mean ± SEM; n = 7). (C) Western blots of extracts of isolated glomeruli from control and LPS-treated mice probed using antibodies against caspase 8 p20, caspase 3 p20 and ß-actin.

 
To further confirm the in vivo coexistence of IL-18 expression and MC apoptosis, RT-PCR and western blot analyses were performed on isolated glomeruli. As shown in Figure 2B, IL-18 mRNA levels in isolated glomeruli from LPS-treated mice were significantly higher than those in control mice (IL-18/GAPDH ratio: 0.35 ± 0.08 vs 0.1 ± 0.04, P < 0.05), consistent with the ISH data (Fig. 2A). In addition, cleavage of both procaspases 8 and 3 were significantly increased in LPS-treated mice compared with control mice (Fig. 2C), providing a signalling mechanism accounting for the enhanced apoptosis induced by LPS (Fig. 2A).

LPS treatment of cultured MCs results in IL-18 expression, but not apoptosis
As shown in Fig. 3A, almost pure MC cultures were established, as most of the cells stained positive for two typical MC biomarkers, {alpha}-SMA and vimetin, but not for E-cadherin, an epithelial cell marker. Preliminary tests showed that 50 µg/ml of LPS were just below the threshold for LPS cytotoxic effects on MCs (data not shown). To evaluate effects of LPS on apoptosis and IL-18 expression in MCs, concentrations between 10 and 50 µg/ml were therefore used.


Figure 3
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 3. DNA fragmentation and IL-18 expression in control and LPS-treated cultured MCs. (A) MCs were positive for {alpha}-SMA and vimetin and negative for the epithelial cell-specific marker, E-cadherin. (B) Agarose gel electrophoresis showing lack of DNA fragmentation over time (0 – 24 hrs) in cultured MCs with or without LPS (50 µg/ml) treatment. Lanes marked (+) are positive controls for apoptosis. (C) RT-PCR showing changes in IL-18 mRNA levels with time in cultured MCs in the presence or absence of LPS (10 µg/ml). The upper panel shows typical agarose gels showing changes in IL-18 and GAPDH mRNAs in LPS-treated MCs and the lower panel line plots for the IL-18/GAPDH ratio over time in control and LPS-treated MCs. (D) Western blots showing changes in IL-18 levels with time in cultured MCs in the presence or absence of LPS (10 µg/ml). The upper panel shows typical blots showing changes in IL-18 and ß-actin levels in LPS-treated MCs; and the lower panel line plots for the IL-18/ß-actin ratio over time in control and LPS-treated MCs. The data in the plots in C and D are shown as the mean ± SEM and the asterisks indicate a significant difference between the groups (n = 7, *P < 0.05).

 
DNA ladder electrophoresis showed that 10–50 µg/ml of LPS did not induce apoptosis (DNA fragmentation) of MCs; results using 50 µg/ml of LPS are shown in Fig. 3B. However, both RT-PCR and western blotting showed that the same concentrations caused significant IL-18 expression over time (Fig. 3C and Fig. 3D). Compared with basal levels, a significant increase in IL-18 mRNA was seen after 12 or 24 h of treatment with 10 µg/ml of LPS, while levels in control MCs remained unchanged (Fig. 3C) (IL-18/GAPDH ratio at 12 and 24 h 0.509 ± 0.012 and 0.414 ± 0.007, respectively, vs 0.164 ± 0.087 and 0.170 ± 0.095 in controls; P < 0.05). Similarly, a marked increase in IL-18 protein levels was seen under the same conditions, while levels in control MCs did not change significantly (Fig. 3D) (IL-18/ß-actin ratio at 12 and 24 h 0.403 ± 0.015 and 0.450 ± 0.034, respectively, compared with undetectable levels in the controls; P < 0.05).

Inability of IL-18 to induce apoptosis in cultured MCs
Using flow cytometry with annexin V as the probe to detect early apoptosis, no significant difference in the percentage of annexin-positive cells was seen between IL-18 (0 – 400 ng/ml) treated and control MCs at 6, 12, or 24 h (P > 0.05); the data for the highest concentration are shown in Fig. 4A. DNA fragmentation, a late event in apoptosis, was also examined by agarose electrophoresis and, again, no apoptosis was seen at 6, 12 or 24 h of IL-18 (0–400 ng/ml) treatment; the data for the highest concentration are shown in Fig. 4B.


Figure 4
View larger version (53K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 4. Lack of effect of IL-18 on apoptosis of cultured MCs. (A) Counting of annexin V-positive cells by flow cytometry over time in control and IL-18-treated (400 ng/ml) MCs. (B) Typical agarose gel showing DNA fragmentation of MCs treated with 400 ng/ml of IL-18 for 0–24 h. Standard ladder DNAs were run in the first lane; the lane marked (+) is a positive control for apoptosis. (C) Levels of procaspase 8 p20 subunit, Bid, Bcl-2, BAX, procaspase 3 p20, or cleaved PARP in cells from both groups. C: control; I: IL-18. (D) Semi-quantitative plots of the levels (test protein/actin ratio) of the indicated proteins. The data are shown as the mean ± SEM (n = 7) and significant differences with P < 0.05.

 
To completely rule out an apoptotic effect of IL-18 on MCs, various apoptotic signalling proteins were evaluated by western blot analyses. The expression of typical apoptotic and anti-apoptotic proteins (p 20 subunit of procaspase 8, Bid, Bcl-2, BAX, p 20 subunit of procaspase 3 and cleaved PARP) in MCs treated with 400 ng/ml of IL-18 for up 24 h did not differ significantly from that in control MCs (P > 0.05); the data for the 24 h treatment are shown in Fig. 4C and D. These results confirm that no apoptotic or anti-apoptotic pathways were activated or suppressed by IL-18.

LPS, but not IL-18, evokes NF-{kappa}B nuclear translocation in cultured MCs
Since nuclear factor {kappa}B (NF-{kappa}B) is a transcriptional regulator that plays a central part in signal transduction by both LPS (Toll-like) receptors and the IL-18 receptor, NF-{kappa}B activation were evaluated in MCs stimulated with LPS or IL-18. No significant difference in NF-{kappa}B nuclear translocation was seen between MCs treated with 400 ng/ml of IL-18 for up to 24 h and control MCs (data not shown), contrasting with the substantial NF-{kappa}B nuclear translocation seen in MCs treated for 6 h with 10 µg/ml of LPS (62 ± 5.5% compared with 11.5 ± 2.1% in controls; P < 0.05) (Fig. 5).


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 5. IF photographs showing NF-{kappa}B nuclear translocation in control, LPS-treated, or IL-18-treated MCs. (A) Typical photographs of IF staining of nuclear NF-{kappa}B in control, LPS-treated, or IL-18-treated MCs. (B) Percentage of cells showing positive nuclear NF-{kappa}B in the three groups. The data are shown as the mean ± SEM (n = 7) and the asterisk shows a significant difference compared with the other two groups (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Statistics
 Results
 Discussion
Acknowledgements
 References
 
In the accelerated-LN mouse model, LPS administration resulted in increased serum auto-antibody levels (Fig. 1D and E), increased immune complex deposition (Fig. 1F), aggravated renal histological profiles (Fig. 2A), and accelerated and exacerbated the clinical syndrome (Fig. 1A–C). These changes can be partly explained by the increased IL-18 expression by MCs induced by LPS (Fig. 2A and Fig. 2B), as IL-18 is a pro-inflammatory cytokine that can recruit and activate neutrophils [30, 31], which, in turn, can result in damage to glomeruli (Fig. 6D). Indeed, marked infiltration of neutrophils was seen around MCs in the LPS-treated mice compared with control mice (Fig. 2A, neutrophils indicated by thin arrows), consistent with the results of previous studies [7–9].


Figure 6
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		FIG. 6. Schematic diagram of the potential actions of LPS and IL-18 on MCs, tubular cells (TCs), and inflammatory cells. (A, B) A direct effect of LPS causing MCs to produce IL-18, (C) a well-known apoptotic autocrine effect of IL-18 on TCs, (D) a well-known effect of IL-18 in activating infiltrating inflammatory cells, (E) an apoptotic autocrine effect of IL-18 and (F) a predicted apoptosis-inducing paracrine effect of IL-18 based on previous reports. MC, mesangial cells; TC, tubular cells; IC, infiltrating inflammatory cells; CL, capillary.

 
IL-18 is produced by many cell types [32], but it is not known if it is expressed in MCs. As shown by ISH in Figure 2, IL-18 expression was detected in MCs and was greatly enhanced by LPS treatment (Fig. 2A and B). Furthermore, the LPS-induced IL-18 expression was a direct, rather than an indirect, effect, as LPS also stimulated the expression of IL-18 mRNA and protein in cultured MCs (Fig. 3). Our in vitro experiment is the first to directly demonstrate that IL-18 is produced in untreated MCs and that this expression is enhanced by LPS. In MCs, LPS might directly control gene expression of IL-18 as the promoter activity upstream of exon 1 in IL-18 gene is regulated by LPS through MyD88-dependent signalling pathway [32–34]. However, our data did not exclude a possibility that other cytokines can mediate the LPS-induced IL-18 up-regulation, which needs further systematical investigations. Besides, the data suggest that an experiment on cultured MCs for IL-18 expression could be confounded by the LPS effect due to LPS impurity in experimental reagents.

The locally produced IL-18 might act in a paracrine and/or an autocrine manner to influence LN progression (Fig. 6). Paracrine roles of IL-18 can be deduced from previous reports, i.e. IL-18 secreted by MCs can boost local inflammation by acting on nearby infiltrating inflammatory cells [30, 31] or can penetrate the capillary to exert an apoptotic paracrine effect on downstream tubular cells [19]. However, whether IL-18 has an autocrine effect on MCs is not known. Microscopically, IL-18 overexpression, hyperplasia and apoptosis of MCs were seen to co-exist in the glomeruli of LPS-accelerated LN mice (Fig. 2A). Excessive apoptosis of MCs can distort the glomerular structure and lead to renal dysfunction, whereas moderate apoptosis can ameliorate MC hyperplasia and maintain the normal glomerular structure [21–23]. Clarifying whether LPS or IL-18 can directly induce MC apoptosis could provide an insight into the pathogenesis of LPS-accelerated LN.

Using primary cultures of MCs from LN-prone mice, the direct effects of LPS and IL-18 on apoptosis of the cells could be precisely evaluated in the absence of the inflammatory cells (Fig. 2A) and auto-antibodies (Fig. 1F) seen in the glomeruli of the LPS-induced accelerated-LN mouse model. To determine the role of LPS in the induction of apoptosis, various concentrations of LPS were used to treat MCs, including the dose of 50 µg/ml, which was 5 times the dose required to induce a significant increase in IL-18 mRNA and protein in MCs (Fig. 3C and Fig. 3D) and is significantly higher than the concentrations required to induce apoptosis in other cells [14–16]. In addition, apoptotic effects of IL-18 on MCs were evaluated by treating the cells with various concentrations (0–400 ng/ml) of IL-18, the highest concentration used being 4 or 40 times higher than that used previously to induce apoptosis of cardiac endothelial cells or tubular cells, respectively [18, 19]. Our in vitro experiments showed that LPS or IL-18 was unable to induce MC apoptosis (Figs 3B and 4) and ruled out the possibility that LPS or IL-18 directly cause the apoptosis of MCs observed in the LPS-treated mice (Fig. 2). These results strongly suggest that, in LPS-exacerbated LN, the enhanced MC apoptosis is unlikely to be caused by a direct effect of circulating LPS and locally produced IL-18, but by other mechanisms [28, 35–38].

A common pathway for signal transduction of LPS and IL-18 after receptor binding is recruitment and activation of MyD88/IRAK, degradation of I{kappa}B, and, finally, induction of NF{kappa}B nuclear translocation [32]. Our data showed that LPS induced marked NF-kB activation in MCs, whereas IL-18 did not (Fig. 5), explaining the LPS-enhanced IL-18 expression (Figs 2 and 3) and inability of IL-18 to induce MC apoptosis (Figs 4A and B). To completely exclude a role of IL-18 in MC apoptosis, our in vitro data showed that IL-18 was unable to activate the extrinsic (procaspase 8), intrinsic (Bcl-2 and Bax) and common (procaspase 3 and PARP) pathways of apoptosis in MCs (Fig. 4C and D), despite the in vivo co-existence of IL-18 overexpression and procaspase activation (Fig. 2B and C).

Taken together, our results demonstrate that LPS enhanced IL-18 expression in MCs both in vivo and in vitro, but neither LPS nor IL-18 directly induced apoptosis in MCs isolated from LN-prone mice. We suggest that the MC-produced IL-18 could act like a paracrine cytokine to exacerbate glomerular inflammation and is probably involved in the apoptosis of the downstream tubular cells, but cannot act as an autocrine cytokine to influence MC survival. We infer that LPS-induced IL-18 up-regulation in MCs might be a mechanism, operating locally in the kidney, responsible for LPS-accelerated LN.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Statistics
 Results
 Discussion
 Acknowledgements
References
 
This study was supported by a grant from the Ministry of Economy (95-EC-17-A-20-S1-028), Taiwan, Republic of China.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Statistics
 Results
 Discussion
 Acknowledgements
 References
 

  1. Tang S, Lui SL, Lai KN. Pathogenesis of lupus nephritis: an update. Nephrology (Carlton) (2005) 10:174–9.[CrossRef][Medline]
  2. Mok CC. Prognostic factors in lupus nephritis. Lupus (2005) 14:39–44.[Abstract/Free Full Text]
  3. Tomita H, Yamada M, Sekigawa I, Yoshiike T, Iida N, Hashimoto H. Systemic lupus erythematosus-like autoimmune abnormalities induced by bacterial infection. Clin Exp Rheumatol (2003) 21:497–9.[Web of Science][Medline]
  4. Gladman DD, Hussain F, Ibanez D, Urowitz MB. The nature and outcome of infection in systemic lupus erythematosus. Lupus (2002) 11:234–9.[Abstract/Free Full Text]
  5. Oh HM, Chng HH, Boey ML, Feng PH. Infections in systemic lupus erythematosus. Singapore Med J (1993) 34:406–8.[Medline]
  6. Janwityanuchit S, Totemchokchyakarn K, Krachangwongchai K, Vatanasuk M. Infection in systemic lupus erythematosus. J Med Assoc Thai (1993) 76:542–8.[Medline]
  7. Cavallo T, Granholm NA. Bacterial lipopolysaccharide transforms mesangial into proliferative lupus nephritis without interfering with processing of pathogenic immune complexes in NZB/W mice. Am J Pathol (1990) 137:971–8.[Abstract]
  8. Cavallo T, Granholm NA. Lipopolysaccharide from gram-negative bacteria enhances polyclonal B cell activation and exacerbates nephritis in MRL/lpr mice. Clin Exp Immunol (1990) 82:515–21.[Web of Science][Medline]
  9. Granholm NA, Cavallo T. Bacterial lipopolysaccharide enhances deposition of immune complexes and exacerbates nephritis in BXSB lupus-prone mice. Clin Exp Immunol (1991) 85:270–7.[Web of Science][Medline]
  10. Baran D, Vendeville B, Ogborn M, Katz N. Cell adhesion molecule expression in murine lupus-like nephritis induced by lipopolysaccharide. Nephron (2000) 84:167–76.[CrossRef][Web of Science][Medline]
  11. Kusner DJ, Luebbers EL, Nowinski RJ, Konieczkowski M, King CH, Sedor JR. Cytokine- and LPS-induced synthesis of interleukin-8 from human mesangial cells. Kidney Int (1991) 39:1240–8.[Web of Science][Medline]
  12. Fouqueray B, Boutard V, Philippe C, et al. Mesangial cell-derived interleukin-10 modulates mesangial cell response to lipopolysaccharide. Am J Pathol (1995) 147:176–82.[Abstract]
  13. Calvani N, Richards HB, Tucci M, Pannarale G, Silvestris F. Up-regulation of IL-18 and predominance of a Th1 immune response is a hallmark of lupus nephritis. Clin Exp Immunol (2004) 138:171–8.[CrossRef][Web of Science][Medline]
  14. Song Y, Shi Y, Harken AH, Meng X. Induction of type II alveolar epithelial cells apoptosis in mouse by lipopolysaccharide does not require TNF-alpha. Chin Med J (Engl) (2003) 116:625–9.[Medline]
  15. Bannerman DD, Goldblum SE. Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis. Am J Physiol Lung Cell Mol Physiol (2003) 284:L899–914.[Abstract/Free Full Text]
  16. Jo SK, Cha DR, Cho WY, et al. Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells. Nephron (2002) 91:406–15.[CrossRef][Web of Science][Medline]
  17. Huang YH, Chang AY, Huang CM, Huang SW, Chan SH. Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells. Proteomics (2002) 2:1220–8.[CrossRef][Web of Science][Medline]
  18. Chandrasekar B, Vemula K, Surabhi RM, et al. Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death. J Biol Chem (2004) 279:20221–33.[Abstract/Free Full Text]
  19. Faust J, Menke J, Kriegsmann J, et al. Correlation of renal tubular epithelial cell-derived interleukin-18 up-regulation with disease activity in MRL-Faslpr mice with autoimmune lupus nephritis. Arthritis Rheum (2002) 46:3083–95.[CrossRef][Web of Science][Medline]
  20. Chandrasekar B, Valente AJ, Freeman GL, Mahimainathan L, Mummidi S. Interleukin-18 induces human cardiac endothelial cell death via a novel signaling pathway involving NF-kappaB-dependent PTEN activation. Biochem Biophys Res Commun (2006) 339:956–63.[CrossRef][Web of Science][Medline]
  21. Shimizu A, Kitamura H, Masuda Y, Ishizaki M, Sugisaki Y, Yamanaka N. Apoptosis in the repair process of experimental proliferative glomerulonephritis. Kidney Int (1995) 47:114–21.[Web of Science][Medline]
  22. Soto H, Mosquera J, Rodriguez-Iturbe B, Henriquez La Roche C, Pinto A. Apoptosis in proliferative glomerulonephritis: decreased apoptosis expression in lupus nephritis. Nephrol Dial Transplant (1997) 12:273–80.[Abstract/Free Full Text]
  23. Baker AJ, Mooney A, Hughes J, Lombardi D, Johnson RJ, Savill J. Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis. J Clin Invest (1994) 94:2105–16.[Web of Science][Medline]
  24. Chen A, Sheu LF, Ding SL, Lee WH. Monomeric immunoglobulin A protects glomerulus against polymeric immunoglobulin A immune complex in experimental immunoglobulin A nephropathy. Lab Invest (1996) 74:737–46.[Web of Science]
  25. Kreisberg JI, Karnovsky MJ. Glomerular cells in culture. Kidney Int (1983) 23:439–47.[Web of Science][Medline]
  26. Shui HA, Ka SM, Lin JC, et al. Fibronectin in blood invokes the development of focal segmental glomerulosclerosis in mouse model. Nephrol Dial Transplant (2006) 21:1794–802.[Abstract/Free Full Text]
  27. Groggel GC, Marinides GN, Hovingh P, Hammond E, Linker A. Inhibition of rat mesangial cell growth by heparan sulfate. Am J Physiol (1990) 258:F259–65.[Web of Science][Medline]
  28. Sugiyama H, Kashihara N, Makino H, Yamasaki Y, Ota Z. Reactive oxygen species induce apoptosis in cultured human mesangial cells. J Am Soc Nephrol (1996) 7:2357–63.[Abstract]
  29. Ka SM, Rifai A, Chen JH, et al. Glomerular crescent-related biomarkers in a murine model of chronic graft versus host disease. Nephrol Dial Transplant (2006) 21:288–98.[Abstract/Free Full Text]
  30. Leung BP, Culshaw S, Gracie JA, et al. A role for IL-18 in neutrophil activation. J Immunol (2001) 167:2879–86.[Abstract/Free Full Text]
  31. Canetti CA, Leung BP, Culshaw S, McInnes IB, Cunha FQ, Liew FY. IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4. J Immunol (2003) 171:1009–15.[Abstract/Free Full Text]
  32. Gracie JA, Robertson SE, McInnes IB. Interleukin-18. J Leukoc Biol (2003) 73:213–24.[Abstract/Free Full Text]
  33. Tone M, Thompson SA, Tone Y, Fairchild PJ, Waldmann H. Regulation of IL-18 (IFN-gamma-inducing factor) gene expression. J Immunol (1997) 159:6156–63.[Abstract]
  34. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol (2001) 19:423–74.[CrossRef][Web of Science][Medline]
  35. Tsai CY, Yu CL, Wu TH, et al. Polyclonal anticardiolipin antibodies purified from sera of patients with active systemic lupus erythematosus induce apoptosis of the cultured glomerular mesangial cells. Scand J Rheumatol (2000) 29:370–9.[CrossRef][Web of Science][Medline]
  36. Tsai CY, Wu TH, Sun KH, Liao TS, Lin WM, Yu CL. Polyclonal IgG anti-dsDNA antibodies exert cytotoxic effect on cultured rat mesangial cells by binding to cell membrane and augmenting apoptosis. Scand J Rheumatol (1993) 22:162–71.[Web of Science][Medline]
  37. Inoue CN. LPA as a determinant of mesangial growth and apoptosis. Semin Nephrol (2002) 22:415–22.[CrossRef][Web of Science][Medline]
  38. Nishizawa Y, Fukai F, Natori Y, Kato R, Tanuma S, Katayama T. Mesangial cell apoptosis induced by a fibronectin fragment. Apoptosis (1998) 3:407–12.[CrossRef][Web of Science][Medline]
Submitted 3 January 2007; revised version accepted 13 April 2007.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
46/8/1277    most recent
kem136v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Shui, H.-A.
Right arrow Articles by Chen, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shui, H.-A.
Right arrow Articles by Chen, A.
Related Collections
Right arrow Systemic Lupus Erythematosus and Autoimmunity
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?