MATERIALS AND METHODS

Animals: Specific pathogen-free male Wistar rats (200- 250 g), purchased from Harlan (outbred strain, Zeist, The Netherlands), were used in this study. The rats received a standard diet and were housed under standard laboratory conditions. The study as presented was approved by the Local Committee for Care and Use of Laboratory Animals and was performed according to strict governmental and international guidelines on animal experimentation.

Production, purification and affinity test of monoclonal anti-Thy 1 antibody: Production of anti-Thy 1 antibody was performed according to Bagchus et al. (1986). Briefly, monoclonal antibody (MAb) against Thy 1 (ER4) was produced in vitro by mouse hybridoma cells. IgG was collected from culture media and subsequently purified using HiTrapTM protein G column (Amersham Pharmacia Biotech AB, Upsala, Sweden). The specificity of this antibody was confirmed by immunohistochemical staining of mesangial cells in healthy rat kidneys.

Induction of glomerulonephritis in rats: To induce acute glomerulonephritis in rats, animals received a single i.v. dose of anti-Thy 1 IgG (5 mg kg^-1) via the penile vein, under anaesthesia with 40% O₂:60% N₂O combined with 0.5% isoflurane.

Experimental design: Six rats per group were used in this study. To examine the acute effects of IL-10, a single i.v. dose (8 μg kg^-1) of this cytokine (Peprotech, UK) with a specific activity of about 5x105 unit mg^-1, was administered to the rats one hour after anti-Thy 1 injection. Control rats received only vehicle (PBS). Subsequently, rats were housed individually in metabolic cages to collect the urine during the experiment. Twenty four hours after anti-Thy 1 administration, rats were sacrificed and samples of excreted urine, serum and kidneys were collected.

Immunohistochemical analysis: Since the monoclonal anti-Thy 1 antibody was detectable by a RAMPO/AEC staining in all glomeruli, 24 h after its administration, all primary antibodies used in this study for immunohistochemical analysis were polyclonal antibodies.

Detection of macrophage and neutrophil infiltrating the glomeruli by CD14 staining: Macrophage and neutrophil infiltration into glomeruli was determined by glomerular CD14 expression. CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein found on the surface of neutrophils and macrophages and can serve as a marker for these cell types (Haziot et al., 1993). The glomerular expression of CD14 was performed with a standard indirect immunostaining method, with amplification. Briefly, cryosections (5 μm) of kidney tissue were fixed in aceton. A goat polyclonal anti-CD14 (1:40, Santacruz Biotechnology, CA, USA) was applied as a primary antibody. A horseradish peroxidase (HRP)-conjugated rabbit polyclonal anti-goat (RAGPO 1:50, DAKO, Glostrup, Denmark) was used as a secondary antibody. To amplify the signal, a horseradish peroxidase (HRP)-conjugated goat polyclonal anti-rabbit (GARPO 1:50, DAKO) was subsequently added. The reaction was visualized with 3-amino-9-ethylcarbazole (AEC, Sigma, St.Louis, USA). Semiquantitative evaluation of the staining for CD14 was performed by scoring of the average of the staining in 40 glomeruli per kidney section per rat using the following score: 0 (no staining within glomeruli), 1 (1 - 25%), 2 (26 – 50%) and 3 (> 50%) of total area/glomerulus.

Determination of glomerular ICAM-1 and MMP-13: The expression of glomerular ICAM-1 and MMP-13 was also determined on cryosections of kidney tissue with standard immunostaining methods with amplification, as described for the CD14 staining. Antibodies used to detect these proteins were respectively goat polyclonal anti-ICAM-1 (1:40, Santacruz Biotechnology, CA, USA) and anti-MMP-13 (1:40, Santacruz Biotechnology, CA, USA). Semiquantitative evaluation of the staining was performed in 40 glomeruli per kidney section per rat with the same score procedure as used for CD14.

Determination of nitric oxide (NOx) in serum: Nitric oxide was determined in serum samples with a standard Griess reaction method (Sen and Donaldson, 1978). The calculation of NOx levels was corrected to endogenous nitrite in the serum which was determined in each sample in a parallel reaction in which the enzymatic nitrate reduction step was omitted.

Determination of urinary protein excretion: Total protein was determined from 24 h-excreted urine samples using a standard Bradford method according to manufacturer’s instruction (Biorad kit protein assay, Biorad, Hercules, CA, USA).

Determination of the mRNA levels for TNF-α, IL-1β, MCP-1 and CD14
mRNA isolation from renal cortex: Total RNA was extracted from tissue samples of the renal cortex with a mini column system according to the manufacturer’s instruction (RNeasy^® mini kit, Qiagen Sciencer, Maryland USA). RNA concentrations in all samples were determined with NanoDrop^® (ND-1000 UV-Vis spectrophotometer, NanoDrop Technology, USA). Integrity of mRNA samples was checked by electrophoresis on a 2% agarose gel and the absence of DNA from the samples was verified by performing a PCR on the mRNA samples using primers for glyceraldehyde-3- phosphate dehydrogenase (GAPDH) while omitting the reverse-transcriptase step. cDNA was prepared from 1 μg of total RNA in 25 μL with RT-PCR method according to the manufacturer’s method (Promega Inc., USA).

Quantitative real-time RT-PCR (QRT-PCR) for TNF-α, IL-1β, MCP-1 and CD14 genes: Quantitative real-time RT-PCR was performed with a high-throughput real-time PCR system (ABI 7900HT sequence Detection System, Applied Biosystems, CA, USA). The PCR mixture (20 μL) contained 1.25 μL cDNA, primers (1 μM concentration of each primer, Table 1) and 2x SyberGreen master mix (Applied Biosystem). An initial denaturing step at 95°C for 10 min was followed by 40 cycles of 95°C for 15 sec, 56°C for 15 sec and 72°C (a measuring step) for 40 sec. Each measurement was performed in three replicates. A single product was confirmed by checking the dissociation curve at the end of the PCR reaction. Data were analysed with the SDS software 2.1 (Applied Biosystems). The gene expressions were normalized to the signal of the house keeping gene β-actin.

Table 1:	Rat oligonucleotide primers used for the analysis of genes by quantitative real- time RT-PCR

Statistical analysis: Data were presented as Mean±SD. All data were subjected to an unpaired, two-tailed distribution student t-test (Microsoft Office excel 2007). Differences were considered significant at p<0.05.

RESULTS

Effect of IL-10 treatment on the inflammatory responses in anti-Thy 1-induced GN: We examined the consequence of IL-10 treatment on inflammatory responses such as infiltration of inflammatory cells, expressions of adhesion molecules and matrix metalloproteinases, NOx levels, genes expression of TNF-α, IL-1β, CD14 and MCP-1 and on glomerular integrity reflected by proteinuria.

Effect of IL-10 treatment on the intraglomerular infiltration of inflammatory cells: Detection of macrophage and neutrophil infiltration into glomeruli was performed by CD14 staining (Fig. 1A, B). As shown in Fig. 1A, CD14 was not expressed by resident glomerular cells. Twenty four hours after anti-Thy 1 administration, this protein was markedly upregulated in the glomeruli. CD14 expression in inflamed glomeruli was distributed throughout the glomeruli and occasionally expressed in the vascular pole. Staining was also detected to a lesser extent in the interstitium of kidneys. The expression of anti-Thy 1-induced CD14 was clearly reduced in IL 10-treated nephritic group. To quantify the effectivity of IL-10 on the reduction of this parameter, we used a score index as described in materials and methods section. According to this system, the effect of IL-10 on the glomerular CD14 expression, presented in Fig. 1B, revealed a reduction in score index from 2.5±0.4 to 1.4±0.2 (p<0.05).

Effect of IL-10 on the protein expression of glomerular ICAM-1 and MMP-13: The immunostaining for glomerular ICAM-1 is shown in Fig. 2A. As can be seen, ICAM-1 was constitutively expressed in renal vascular endothelium and occasionally in the glomeruli at a very low level. A strong induction of the expression of glomerular, tubular and renal interstitial ICAM-1 was already detected 24 h after administration of anti-Thy 1 which is also reported by others (Arrizabalaga et al., 1997; Chiang et al., 2006; Ikeda et al., 1996). The expression of ICAM-1 in particular in the mesangial area and in renal tubuli was clearly inhibited by IL-10 treatment. Semiquantitative evaluation of the effect of IL-10 on this expression, presented in Fig. 2B, revealed a reduction in score index from 1.9±0.3 to 1.3±0.2 (p<0.05).

Fig. 1:

The effect of IL-10 on the intraglomerular macrophage and neutrophil recruitment. (Fig. 1A) Infiltrated macrophages and neutrophils were detected by CD14 staining. (a) CD14 is not constitutively expressed in the glomeruli. (b) This expression was markedly induced after anti-Thy 1 administration, in particular around the vascular pole (arrow). (c) Treatment with IL-10 significantly reduced the staining (Fig. 1B) Using a semiquantitative scoring system, 40 glomeruli per rat and 6 rats per group were analyzed according to the following score: 0 (no staining within glomeruli), 1 (1-25%), 2 (26-50%) and 3 (>50%) of total area/glomerulus. Original magnification: 200x. *p<0.05

In addition to the glomerular ICAM-1 staining, glomerular MMP-13 expression was determined. As shown in Fig. 3A, in normal rats, MMP-13 was highly expressed around renal blood vessels and occasionally at a very low level in the glomeruli whereas the tubuli were negative. The glomerular and tubular MMP-13 staining was however strongly enhanced 24 h after administration of anti-Thy 1 antibody. Expression of this protease was found around capillary loops of glomeruli, brush borders of distal and proximal tubuli and around renal blood vessels. Similar to the glomerular ICAM-1 staining, a reduced glomerular and tubular MMP-13 staining was seen in the IL-10-treated nephritic group. Semiquantitative evaluation of kidney sections revealed that IL-10 reduced the score index of this parameter from 2.1±0.5 to 1.4±0.5 (p<0.05, Fig. 3B).

Fig. 2:

The effect of IL-10 on glomerular ICAM-1 staining. (A) Immunohistochemical staining of ICAM-1 in kidneys. (a) ICAM-1 was expressed in the glomeruli at a very low level. (b) This expression was markedly induced after anti-Thy 1 administration. (c) Treatment with IL-10 significantly reduced the staining. (B) Using a semiquantitative scoring system, 40 glomeruli per rat and 6 rats per group were analyzed according to the following score: 0 (no staining within glomeruli), 1 (1-25%), 2 (26-50%) and 3 (>50%) of total area/glomerulus. Original magnification: 200x. *p<0.05

Effect of IL-10 on serum levels of Nox: NOx is one of products of activated macrophages in response to various stimuli and is subsequently released into the circulation.

Fig. 3:

The effect of IL-10 on the glomerular MMP-13. (A) Immunohistochemical staining of MMP-13 in kidneys. (a) MMP-13 was expressed in the glomeruli at a very low level and strongly around renal blood vessels (arrow). (b) This expression was markedly induced after anti-Thy 1 administration and expressed in the vascular loops of glomeruli and in the tubuli (arrow). (c) Treatment with IL-10 significantly reduced the staining. (B) Using a semiquantitative scoring system, 40 glomeruli per rat and 6 rats per group were analyzed according to the following score: 0 (no staining within glomeruli), 1 (1-25%), 2 (26-50%) and 3 (>50%) of total area/glomerulus. The original magnification: 200x. *p<0.05

As shown in Fig. 4A and B, the average serum NOx level in normal rats was 7.3±1.3 μM, whereas 24 h after anti-Thy 1 injection, serum NOx levels rose to 23.9±10.3 μM. In IL-10-treated nephritic rats, serum NOx levels felt to 16.3±6.7 μM. This difference was not significant compared to untreated nephritic rats.

The effect of IL-10 on the urinary protein excretion: Administration of anti-Thy 1 antibody to the rats resulted in a rapid onset of proteinuria within 24 h (113±51.0 mg/24 h) compared to normal rats (21.0±9.2 mg/24 h). Urinary protein excretion in IL-10-treated nephritic rats was 76.6±59.4 (Fig. 5). This difference was not statistically significant as compared to untreated nephritic rats and also not significantly different from normal rats.

Fig. 4:	The serum NOx levels after disease induction with anti-Thy 1 IgG. The serum NOx levels significantly increased in nephritic rats as compared to normal rats. *p<0.05

Fig. 5:	The urinary protein measurement, showing the development of proteinuria (mg/24 h) in anti-Thy 1 induced nephritic rats. *p<0.05

Effect of IL-10 treatment on the genes expression of TNF-α, IL-1β, MCP-1 and CD14: We examined the mRNA levels for inflammatory parameters using quantitative real-time PCR techniques. mRNA levels for TNF-α and IL-1β were measured to assess proinflammatory activity, mRNA levels for CD14 were used as a marker for neutrophil and macrophage influx and MCP-1 mRNA levels were measured to assess chemotactic activity. β-actin expression was used as the house keeping gene. As shown in Fig. 6, all genes examined were significantly upregulated as compared to normal rats, in particular MCP-1 was substantially increased. However, in IL-10-treated nephritic rats, expression of all these genes did not significantly change in comparison with the control nephritic group.

Fig. 6:	A quantitative real-time PCR of TNF-α, IL-1β, CD14 and MCP-1. A single shot injection of anti-Thy 1 IgG increased the mRNA levels for TNF-α, IL-1β, CD14 and markedly increased MCP-1 gene expression level. The expression of these genes was not different between control GN and IL-10-treated groups. *p<0.05 as compared to normal

DISCUSSION

IL-10 is an anti-inflammatory cytokine which suppresses the T helper 1 immune response and downregulates macrophage and monocyte activities. Because of its potent anti-inflammatory properties, IL-10 is now being tested in the therapy of inflammatory diseases like inflammatory bowel disease, rheumatoid arthritis and psoriasis (Asadullah et al., 2003). In addition, several studies reported that IL-10 effectively prevented the development of glomerular damage both in acute and chronic nephritis models (Schneider et al., 2004; Scholz et al., 2008; Blenman et al., 2006; Kitching et al., 2002; Ogando et al., 2004; Cassatella et al., 1993). In a previous study, we found rapid accumulation of IL-10 in the kidney (30% of the injected dose within 10 min) and intraglomerular IL-10 receptor expression (Rachmawati et al., 2004). We therefore considered the possibility that IL-10 might be applicable for the treatment of in particular kidney diseases. However, the very short plasma half-life of approximately 2 min would not favour an IL-10-based therapy for chronic diseases. This rapid clearance of IL-10 from the plasma, prompted the question whether IL-10 might have a pharmacological effects after 24 h. For that reason we examined the effect of a single dose of IL-10 after 24 h in a model of acute GN.

Anti-Thy 1 nephritis is caused by the binding of Thy 1 IgG to the mesangial cell surface, which results in complement-dependent glomerular damage. Within hours, a destruction of mesangial cells occurs which is accompanied by acute inflammatory reactions. As we presented in this study, mRNA levels for IL-1β, TNF-α, CD14 and MCP-1 in the kidney clearly increase in response to this anti-Thy 1 deposition. mRNA levels for IL-1β, TNF-α and CD14 in nephritic rats were moderately increased at 24 h, while mRNA level for MCP-1 was very strongly upregulated at this time point (Fig. 6). The increase of these proinflammatory factors was accompanied by a marked intraglomerular recruitment of macrophages and neutrophils, as reflected by the CD14 staining (Fig. 1A, B). We stained CD14, one of macrophage and neutrophil markers, to identify the glomerular infiltration of these cells. Because, especially after 24 h of the disease induction, MAb anti-Thy 1 antibody was still clearly present, utilization of all monoclonal antibodies to identify the cells was excluded.

In this report, we demonstrate that a single iv injection of IL-10 significantly attenuated CD14 and ICAM-1 expression, macrophage and neutrophil recruitment and MMP-13 expression. Proteinuria was reduced to a level intermediate between diseased and normal rats (not significantly changed compared to either group). So, the effect of IL-10 on proteinuria is yet inconclusive. In contrast to all this, we did not find any difference on the gene expression of CD14, IL-1β, TNF-α and MCP-1 between IL-10-treated and control GN groups. In particular for CD14, IL-10 attenuated this parameter at the protein levels but not at the mRNA level. We therefore conclude that the effect of a single injection of this short-lived cytokine is still no after 24 h on many inflammatory parameters but at this time point the effect on the examined gene expression levels is not longer present.

In addition to the inhibition of glomerular ICAM-1 and CD14, a reduced glomerular MMP-13 expression was also noted. MMP-13 is one of matrix metalloproteinases (MMPs) with high collagenolytic activity (Gomis-Ruth, 1996; Manek and Lane, 2000). The expression of this matrix degrading enzyme is associated with inflammatory activity and tissue destruction. It was also found that MMP-2, MMP-3 and MMP-9 were induced in both patients with glomerulonephritis and in rat models of experimental glomerulonephritis, indicating a role for these enzymes in this disease. Accumulating evidence indicates that their expression is regulated by proinflammatory cytokines such as IL-1β, TNF-α, TGF-β and also proinflammatory mediators like nitric oxide (Sanders et al., 2004; Urushihara et al., 2002; Steinmann-Niggli et al., 1998). Moreover, it has been reported that expression of the collagenase MMP-13, correlated with arthritis in patients and in experimental models. To our knowledge, the induction of glomerular MMP-13 in rats with acute glomerulonephritis, reported here, is studied for the first time. MMP-13 is expressed around blood vessels in normal kidneys and in the glomeruli at very low level. Its high expression in anti-Thy 1 nephritic rats indicates a role in the pathogenesis of this disease. The mechanism underlying its upregulation in this model remains unclear, but previous studies showed that persistent accumulation of macrophages in the mesangium area resulted in glomerulosclerosis through expression and activation of MMPs, suggesting that infiltrated macrophages participate in this event. This is supported here by the glomerular CD14 expression.

In view of the effects of IL-10 on macrophage recruitment, we determined serum NOx level as one of the products of activated macrophages. NO plays an important regulatory role in a variety of inflammatory conditions. The generation of NOx in serum was observed after anti-Thy 1 administration, reflecting the development of disease. However, administration of IL-10 did not result in a significant reduction of NOx levels. Reduced macrophage recruitment by IL-10 seemed not to be associated with an effect of treatment on the NOx levels.

In summary, this study demonstrates that despite its short plasma half-life of only 2 min, a single iv dose of IL-10 can suppress inflammatory processes during acute glomerulonephritis induced by anti-Thy 1 after 24 h. A possible anti-inflammatory mechanism underlying this acute effect of IL-10 may occur via inhibition of glomerular ICAM-1 expression, resulting in reduced macrophage recruitment reflected by reduced CD14 staining. The reduced MMP-13 after IL-10 treatment indicates that IL-10 may be beneficial to preserve glomerular integrity. This decrease of inflammatory parameters at the protein levels and the glomeruloprotective effect induced by IL-10 was however not associated with an effect of treatment on the mRNA levels for the parameters examined. The effects of IL-10 demonstrated here call for further evaluation, but may indicate a role for this cytokine as a therapeutic compound during glomerulonephritis. In this framework, various dosage regiments should be investigated at different stages of the disease also using additional animal models. The anti-inflammatory effects of this short-lived cytokine found one day after its administration provide good perspectives for chronic applications.

ACKNOWLEDGMENTS

We are grateful to Dr. W.W. Bakker, Department of Pathology, University Medical Center Groningen, The Netherlands, for kindly providing the ER4 cells. This project was supported by a grant from Islamic Development Bank (IDB), Jeddah, Saudi Arabia.