Inhibition of interleukin-10 by the immunomodulator AS101 reduces mesangial cell proliferation in experimental mesangioproliferative glomerulonephritis: association with dephosphorylation of STAT3.

Mesangial cell (MC) proliferation is essential for the pathogenesis and progression of glomerular disease. Using an acute model of mesangial proliferative glomerulonephritis (Thy1 GN), we show that neutralization of interleukin (IL)-10 greatly ameliorated the disease as expressed by both decreased MC expansion and proteinuria. Treatment with the tellurium compound AS101 (ammonium trichloro(dioxoethylene-o,o')tellurate) resulted in favorable effects provided that the compound was administered 24 h before insult, whereas partial effects were obtained when administered after insult. We identified STAT3 as playing a pivotal role in IL-10-induced MC proliferation in vitro and in vivo. IL-10 activates MC STAT3 in vitro as expressed by its phosphorylation and nuclear translocation. The role of STAT3 in MC proliferation induced by IL-10 was deduced from results showing that IL-10-induced proliferation was abrogated if MC transfected with STAT3 antisense oligonucleotides were used or if cells were incubated with inhibitors of STAT3. AS101 deactivates STAT3 in control but not in MC transfected with IL-10 antisense oligonucleotides. Inactivation of STAT3 prevents reduction of MC proliferation by AS101. We further demonstrate the role of STAT3 in the regulation of cell cycle and survival regulatory proteins by AS101 in MC via inhibition of IL-10. IL-10 increased MC expression of Bcl-2 and Bcl-X1 and simultaneously decreased the levels of p27kip1. These survival factors were decreased by AS101 in a STAT3- and IL-10-dependent manner, whereas p27kip1 was similarly increased. In Thy1 GN, phosphorylated STAT3 in glomerular MC peaked at day 6 and correlated with MC expansion. Neutralization of IL-10 or its inhibition by AS101 abolished phosphorylation of STAT3. This effect positively correlated with amelioration of the disease. These in vitro and in vivo studies indicate that the autocrine MC growth factor IL-10 induces MC proliferation via STAT3. We suggest that IL-10 or its downstream target STAT3 might be therapeutic targets for kidney diseases induced by mesangial proliferation.

Cellular proliferation accompanies a wide variety of renal diseases. This is especially true for proliferative forms of glomerulonephritis (GN) 1 (1). Excessive proliferation of mesangial cells is usually associated with matrix expansion, leading to the development of glomerular sclerosis (2). Mesangial cells proliferate in response to a variety of growth factors and cytokines such as platelet-derived growth factor (PDGF), basic fibroblast growth factor, and interleukin-6 (3)(4)(5). Recently, we showed that IL-10 is a potent mesangial autocrine growth factor that plays a pivotal role in rat MC proliferation in vitro (6). Therefore, its inhibition by the non-toxic tellurium anti-IL-10 compound, ammonium trichloro(dioxoethylene-o,oЈ) tellurate (AS101), extensively decreased proliferation of these cells (6).
IL-10 first recognized for its ability to inhibit activation and effector function of T cells, monocytes and macrophages, is a multifunctional cytokine with diverse effects on a variety of cell types. The principal function of IL-10 appears to be limitation and ultimately termination of inflammatory responses. In addition, IL-10 regulates growth and/or differentiation of B cells, natural killer cells, cytotoxic and helper T cells, mast cells, granulocytes, dendritic cells, keratinocytes, and endothelial cells (7). The best-characterized IL-10 signaling pathway is the JAK/signal transducer and activator of transcription (STAT) system. The IL-10/IL-10R interaction engages the Jak family tyrosine kinases JAK1 and Tyk2 and induces tyrosine phosphorylation and activation of the latent transcription factors STAT3 and STAT1 (8). The STATs comprise a family of functionally related proteins that play key roles in a variety of biological activities such as cell differentiation and proliferation. They exert their activities through cytokine and growth factor receptors and are believed to be involved in determining the biological specificity of specific cytokines on various target tissues (9). The molecular events that underlie cell proliferation are tightly regulated in a cell cycle-dependent manner. The cell division cycle is coordinated by the activation and inactivation of the cyclin-dependent kinases (CDKs) (10). The G 1 to S phase transition in the cell cycle is thought to be controlled by CDKs that are sequentially regulated by cyclins D, E, and A. These are negatively regulated by two distinct families of CDK inhibitors. One of these includes p21 cip , p27 kip1 , and p57 kip2 , which bind and inhibit cyclin D-, E-, and A-dependent kinases (11). STAT3 has been previously reported to play an important role in the G 1 to S phase transition through the up-regulation of cyclins D and A and the concomitant down-regulation of p21 and p27 (12). STAT proteins are latent transcription factors that become activated by phosphorylation of a single tyrosine, which causes STAT proteins to dimerize. STAT dimerization is usually followed by translocation to the nucleus (13). Within the nucleus, STAT proteins recognize and bind to consensus DNA binding sites that represent enhancer sequences for a variety of genes. STAT3 activation has been implicated in the proliferation of cells (14).
The non-toxic immunomodulator AS101 first developed by us has been shown to have beneficial effects in diverse preclinical and clinical studies. Most of its activities have been primarily attributed to the direct inhibition of the anti-inflammatory cytokine IL-10 (15)(16)(17). This immunomodulatory property was found to be crucial for the clinical activities of AS101, demonstrating the protective effects of AS101 in parasite and viralinfected mice models (18), in autoimmune diseases (19), in septic mice (20), and in a variety of tumor models in mice and humans where AS101 had a clear anti-tumoral effect (21)(22)(23). More importantly, AS101 was previously shown to delay the onset of autoimmune manifestations in a murine model of lupus erythematosus, reduce the level of immune complex deposition in the glomeruli, reduce proteinuria, prevent glomerular hypercellularity and mesangial expansion, and reduce the mean glomerular volume of treated mice (19). Furthermore, in a murine model of septic peritonitis, AS101 was recently shown to prevent kidney damage of septic mice (20).
Given our recent findings that IL-10 is a potent mesangial autocrine growth factor and the ability of AS101 to reduce MC proliferation in vitro via inhibition of IL-10, this study primarily aimed to explore the role of each one of them in experimental mesangioproliferative glomerulonephritis. In addition, because it has been recently demonstrated that in the Thy1 model STAT3 is phosphorylated in glomerular mesangial cells (24), this study also aimed to examine whether the beneficial effects of AS101 are associated to the deactivation of STAT3 via IL-10 inhibition. Furthermore, we studied the implications of this activity on the regulation of cell cycle regulatory proteins in vivo.

Animals
Male Wistar rats (8 -10 weeks old, 150 -170 g) were purchased from Harlan Laboratories (Jerusalem, Israel). Animal experiments were performed in accordance with approved institutional protocols and approved by the Institutional Animal Care and Use Committee.
Rats were housed in individual metabolic cages with free access to food and water. The weight of rats in the various groups was similar at day 0 and at day 6 after OX-7 administration. A 24-h urine collection was obtained for determination of protein excretion on days 0, 2, 4, and 6. Following nephrectomy, tissues were fixed in formalin for histology and immunohistochemistry or were further processed for isolation of glomeruli.

Isolation of Glomeruli
Glomeruli were isolated from the renal cortex of rats using the differential sieving method. The purity of glomeruli was Ͼ95% (6).

MC Cultures
Two sources of rat glomerular MC were used for in vitro studies. 1) An immortalized rat mesangial cell line (RMC) was kindly provided by Dr. M. Allenberg (Department of Medicine, Toronto University, Toronto, Canada). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin. 2) Primary rat glomerular mesangial cells of glomeruli from rat kidneys were isolated. RMCs were cultured in DMEM supplemented with 20% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin. RMCs were used between passages 5 and 10.

Clonogenic Assay
Following 24 h of serum starvation, cells were disrupted by repeated aspiration through a 26-gauge needle, counted, and plated at 1000 cells/plate in DMEM supplemented with 10% fetal calf serum and incubated for macroscopic colony formation. Following 4 -5 days of incubation, colonies were fixed with methanol, stained with Giemsa, and counted.

[ 3 H]Thymidine Uptake
RMCs were cultured in flat-bottomed 96-well plates at 1.10 5 cells/ well in medium containing 10% fetal calf serum. After 24 h, cells were growth-arrested for 48 h in medium containing 0.5% fetal calf serum. MCs were then exposed for 24 h to fresh medium without or with 2% fetal calf serum in the presence of specific components. Cells were pulsed with 1 Ci/ml [ 3 H]thymidine for the last 24 h.

Immunoprecipitation and Western Blot Analysis
Total, cytoplasmic, or nuclear cell extracts and immunoprecipitation were performed as described previously (22). P27 kip1 , Bcl-2, and Bcl-x1 antibodies were from Santa Cruz Biotechnology with the exception of STAT3 and pSTAT3 (New England Biolabs, Beverly, MA).
For assessment of transfection efficiency, cell lysates have been prepared and analyzed by Western blot analysis for STAT3 protein expression. Polycationic transfection reagent (LipofectAMINE, Invitrogen) was used to facilitate uptake of ODN according to the protocol recommended by the manufacturers.

Histopathologic Examination
Light Microscopy-Resected kidneys were cut by a coronal section through the midportion of the kidney. One-half was fixed in 10% buffered formalin, paraffin blocks were prepared, and several 3-10 M sections were cut from each block and stained with hematoxylin-eosin and periodic acid Schiff stains.
Morphometry-Glomerular volumes were evaluated using stereologic methods (24). The mean glomerular tuft area (A gl ) was estimated using a digitizing tablet by tracing the outline of the glomerular tuft of 30 non-globally sclerotic glomeruli from each animal on periodic acid Schiff-stained sections. A point-counting method was utilized to estimate the percentage of the glomerular tuft occupied by mesangium or sclerosis (the mesangial fractional volume Vv mes ) and the percentage of the glomerular tuft occupied by nuclei (the nuclear fractional volume Vv nucl ).
Calculations-Mean glomerular tuft volume (V gl ) was calculated as: V gl ϭ (␤/) ϫ (A gl ) 3/2 , where ␤ ϭ 1.38 is the shape coefficient for spheres and ϭ 1.1 is the size distribution coefficient. The mean glomerular mesangial volume (V mes ) was calculated as: V mes ϭ Vv mes ϫ V gl .

Immunohistochemistry
Parafin sections were prepared and stained with mouse anti-human ␣-smooth muscle actin (␣SMA) antibody (Zymed Laboratories Inc., San Francisco, CA) and the avidin-biotin complex-staining kit. Diaminobenzidine tetrahydrochloride was used as substrate. Each slide was counterstained with hematoxylin. Controls included omitting the primary antibody and its substitution with mouse IgG.

Immunofluorescent Double and Single Staining
Double immunostaining for ␣SMA and phospho-STAT3 was performed on snap-frozen cryostat sections (4 m) using the indirect immunofluorescence procedure. Both primary antibodies (␣SMA (SIGMA) and pSTAT3) were incubated overnight at 4°C followed by the incubation of Texas Red-conjugated anti-rabbit IgG (for pSTAT3) and FITCconjugated anti-mouse IgG (for ␣SMA) (Jackson ImmunoResearch Laboratories, West Grove, PA).
To examine the glomerular binding of the Thy1 antibody and local complement activation, detection of mouse IgG and rat C3 in glomeruli was carried out on frozen cryostat sections using direct immunofluorescence staining with either FITC-conjugated anti-mouse IgG (Jackson ImmunoResearch) or FITC-conjugated anti-rat C3 (ICN Radiochemicals Inc., Costa Mesa, CA)

Statistical Analysis
Data are presented as mean Ϯ S.E. Multiple comparisons were performed with the Kruskal Wallis analysis of variance. The Mann-Whitney U test was used for comparisons between each of the two groups. For comparisons within the groups, Wilcoxon-signed Rank test was performed. Student's two-tail t Ͻ0.05 was considered significant.

Inhibition of IL-10 Attenuates Glomerular Mesangial Cell
Expansion in Vivo-We have previously shown that IL-10 is a potent mesangial autocrine growth factor in vitro whose inhibition by AS101 dramatically reduces MC proliferation (6). We have extended these in vitro observations to an in vivo model of glomerulonephritis. We used an acute model of mesangial proliferative glomerulonephritis known as Thy1 GN. Activated mesangial cell expansion, as illustrated by positive staining for ␣SMA, was compared in glomeruli of the different treatment groups at day 6, which represents the peak of mesangial cell proliferation. Fig. 1A shows a prompt abundance of ␣SMAexpressing cytoplasm in the control anti-Thy1 group (b 1 ), whereas no staining was seen in glomeruli of control healthy rats (a), implying a substantial increase in activated mesangial cells in glomeruli of diseased rats. The results show that treatment with the anti-IL-10 compound AS101 starting 24 h before anti-Thy1 administration greatly ameliorated the disease, decreasing the level of activated mesangial cell expansion to that seen in normal rats (c and c 1 ). Nevertheless, treatment with AS101 24 h after insult did not affect considerably glomerular pathology (d and d 1 ). The results depicted in Fig. 1A illustrate the extensive prevention of mesangial cell activation and proliferation by anti-IL-10 therapy (f and f 1 and g and g 1 ). Compared with the vast expansion of mesangial cells in glomeruli of isotype-matched-treated rats (e and e 1 ), no staining of ␣SMA appeared in glomeruli of rats in which IL-10 was neutralized either 24 h before (f 1 ) or 24 h after (g 1 ) insult. Evaluation of IL-10 protein expression in lysates of isolated glomeruli dissected from rats at day 6 revealed high abundance of this cytokine in control glomeruli of anti-Thy1-treated rats as compared with healthy rats (Fig. 1B). IL-10 protein expression considerably decreased in glomeruli of both anti-IL-10 and AS101-treated rats (Fig. 1B). Nevertheless, inhibition by IL-10 occurred earlier in glomeruli of rats treated with AS101 before insult compared with rats treated with the compound 24 h after FIG. 1. A, hematoxylin-eosin staining (a-g) and immunostaining for ␣SMA (a 1g 1 ) in Thy1 glomerulonephritis on day 6. a and a 1 , control healthy rats treated daily with PBS. b and b 1 , control anti-Thy1treated rats. c and c 1 , AS101-treated rats starting 24 h before anti-Thy1. d and d 1 , AS101-treated rats starting 24 h after anti-Thy1. e and e 1 , control goat IgG-treated rats. f and f 1 , goat anti-IL-10 IgG-treated rats starting 24 h before anti-Thy1. g and g 1 , goat anti-IL-10 IgG-treated rats starting 24 h after anti-Thy1 (magnification, ϫ400). B, IL-10 protein expression in isolated glomeruli of rats with Thy1 glomerulonephritis on day 6. Total cell lysates were prepared from isolated glomeruli of kidneys from rats of the different treatment groups. The results represent one representative experiment of three performed.
Thy1. There was no difference in the intensity or distribution of glomerular immunostaining (day 2) for mouse IgG or rat C3 in rats that were given anti-IL-10 antibodies or AS101, either before or after Thy1 administration (data not shown). These results preclude the possibility that reduction of MC expansion by either AS101 or anti-IL-10 was the result of their interference with binding of the Thy1 antibody or complement activation.
Assessment of Proteinuria in Vivo-To assess whether resolution of activated mesangial cells expansion in treated rats is also reflected by decreased proteinuria, 24-h urinary protein excretion was evaluated in all of the treatment groups at days 0, 2, 4, and 6. Fig. 2a shows that the anti-Thy1 group developed glomerulonephritis as expressed by proteinuria, the levels of which increased from 3.2 Ϯ 0.6 mg/24 h on day 0 to 119.4 Ϯ 9.3 mg/24 h at day 6 (p Ͻ 0.01). These day 6 levels also significantly differed from proteinuria of control rats. Treatment with AS101 starting 24 h before insult significantly ameliorated proteinuria to levels not different from the control group but significantly lower than the anti-Thy1 group throughout the study (p Ͻ 0.01). Nevertheless, treatment with AS101 after anti-Thy1 administration resulted in a significant increase versus day 0 (2.7 Ϯ 0.6 to 35.5 Ϯ 5.4 mg/24 h at day 6; p Ͻ 0.01). However, this excretion rate at both days 4 and 6 was significantly lower than that of the anti-Thy1 group (p Ͻ 0.001). Neutralization of IL-10 resulted in an extensive and significant decrease in proteinuria compared with that of the anti-Thy1 group (p Ͻ 0.01) if neutralizing Abs were injected either before or after Thy1. Proteinuria of the isotype-matched control group did not differ significantly from that of the anti-Thy1 group (Fig. 2b).
Morphometric Analysis of Glomeruli from AS101 and Anti-IL-10-treated Rats-Morphometric analysis of glomeruli is detailed in Table I. Mean glomerular and mean mesangial volumes increased significantly in the Thy1 rats compared with controls (Table I). Cells proliferated significantly as represented by the significant expansion of nuclear volume in the Thy1 rats. Treatment with AS101 before anti-Thy1 administration reduced glomerular, mesangial, and nuclear volumes significantly to levels that were not different from those in controls. Treatment with AS101 after anti-Thy1 administration had no significant effect on any of these parameters (Table I, footnote a). Treatment with anti-IL-10 antibodies before as well as 24 h after Thy1 administration reduced glomerular, mesangial, and nuclear volumes significantly compared with their respective controls (Table I,  AS101 Decreases Tyrosine Phosphorylation and Nuclear Translocation of STAT3 via Inhibition of IL-10 -High expression of phosphorylated STAT3 in glomerular mesangial cells, the level of phosphorylation correlating with peak of mesangial cell proliferation, has been recently reported in a model of Thy1 GN (25). Because IL-10 activates STAT3 in a variety of cells (8), we first examined whether IL-10 activates mesangial STAT3 in vitro and whether its inhibition by AS101 will decrease this activation. We measured STAT3 tyrosine phosphorylation and translocation to the nucleus. Fig. 3a shows that IL-10 at 100 ng/ml increased phosphorylation of STAT3 in starved mesangial cells exposed to this growth factor for 5-15 min. Thereafter, the phosphorylation level significantly decreased. A gradual translocation of phospho-STAT3 to the nucleus was seen in both nuclear extracts immunoblotted with anti-phospho-STAT3 (Fig. 3b) and in mesangial cells stained with this anti-FIG. 2. Urinary protein excretion by rats with Thy1 glomerulonephritis. a, vehicle control (n ϭ 6) rats injected with PBS, ␣Thy1 (n ϭ 8) rats injected with anti-Thy1 Abs, ␣Thy1ϩAS101 before (n ϭ 10) rats injected with AS101 starting 24 h before anti-Thy1 Abs injection, and ␣Thy1ϩAS101 after (n ϭ 9) rats injected with AS101 starting 24 h after anti-Thy1 Abs injection. *, p Ͻ 0.01 compared with ␣Thy1 group on the same day; **, p Ͻ 0.01 compared with ␣Thy1 on day 0; #, ␣Thy1ϩAS101 after compared with vehicle control. b, isotype-matched control (n ϭ 3) rats injected with isotype-matched anti-rat IgG, ␣IL-10 before (n ϭ 3) rats injected with goat anti-rat IL-10 IgG 24 h before injection of anti-Thy1, and ␣IL-10 after (n ϭ 3) rats injected with anti-rat IL-10 IgG 24 h after injection of anti-Thy1. *, p Ͻ 0.05 compared with isotypematched control.
body following incubation of the cells with IL-10 ( Fig. 3c). Incubation of MC in complete medium supplemented with AS101 for 10 -15 min did not affect STAT3 phosphorylation and nuclear translocation (Fig. 4, A and B). However, after long periods of incubation with AS101 (6 -24 h), a gradual decrease in STAT3 phosphorylation was seen (Fig. 4A). This was correlated with a significant decrease in IL-10 expression in treated cells at long (hours) but not at short (minutes) time periods. Thus, AS101 probably does not affect directly STAT3 activity. Nevertheless, by inhibiting IL-10, which is produced at later time points, this activity is significantly decreased. As further confirmation of the IL-10-dependent inhibition of STAT3 phosphorylation by AS101, we show that AS101 did not decrease phosphorylation of STAT3 in mesangial cells transfected with IL-10 antisense oligonucleotides (Fig. 5).
Role of STAT3 in the Inhibition of MC Proliferation by AS101-In these experiments, we first examined whether IL-10-mediated increased proliferation of rat mesangial cells, as previously reported (6), depends on STAT3. IL-10 at 100 ng/ml increased clonogenicity and [ 3 H]tritiated thymidine uptake of rat MC transfected with control oligonucleotides from 51 Ϯ 1.15 to 86 Ϯ 2.33 colonies (p Ͻ 0.01) and from 3,592 Ϯ 40 to 9,759 Ϯ 338 cpm (p Ͻ 0.01), respectively (Fig. 6, A and B). However, this concentration of IL-10 did not increase signifi-cantly the clonogenicity and [ 3 H]thymidine uptake of cells transfected with AS-STAT3 oligonucleotides (Fig. 6, A and B). These results indicate that STAT3 is involved in mesangial cell proliferation induced by IL-10. The role of STAT3 in the inhibition of MC clonogenicity by AS101 could be deduced by the 3 545 Ϯ 32** 312 Ϯ 9** 148 Ϯ 5** a Control group ϭ rats with vehicle (PBS); ␣Thy1 ϭ rats injected with anti-Thy1 Abs; AS101 before ϭ rats injected with AS101 starting 24 h before anti-Thy1 Abs injection; AS101 after ϭ rats injected with AS101 starting 24 h after anti-Thy1 Abs injection.
b Isotype-matched controls ϭ rats injected with anti-Thy1 Abs and isotype-matched anti rat IgG; ␣Thy1ϩanti IL-10 before ϭ rats injected with anti-rat IL-10 Abs 24 h before injection with anti-Thy1 Abs; ␣Thy1ϩanti IL-10 after ϭ rats injected with anti rat IL-10 Abs 24 h after anti-Thy1 Abs injection. fact that no significant inhibition of MC clonogenicity and [ 3 H]thymidine uptake by AS101 was seen when cells transfected with AS-STAT3 oligonucleotides were used (Fig. 6, C and  D). Moreover, using two chemical STAT3 pathway inhibitors, namely piceatannol (JAK1/STAT3 inhibitor) and tyrphostin AG490 (JAK2/STAT3 inhibitor), we found that AS101 does not inhibit proliferation of MC in which STAT3 was inhibited (Fig. 7).
Treatment with AS101 or Anti-IL-10 Antibodies Deactivates Mesangial STAT3 in Vivo-Because AS101-mediated inhibition of mesangial cell proliferation appears to depend on suppression of IL-10-mediated STAT3 activation in vitro, we examined the role of STAT3 in the decreased MC expansion observed in rats treated with AS101 or anti-IL-10 Abs in vivo glomerular proteins collected at days 0, 2, 4, and 6 after Thy1 administration revealed a gradual increase in phosphorylated STAT3, which peaked at day 6 ( Fig. 8A). Treatment with either AS101 or anti-IL-10 antibody 24 h before Thy1 administration totally abrogated glomerular expression of phosphorylated STAT3 on day 6. This was evident by both the decreased pSTAT3 expression in lysates of isolated glomeruli (Fig. 8B) as well as the decreased double-positive staining of pSTAT3 and ␣-smooth muscle actin as presented in Fig. 9. Treatment with anti-IL-10 24 h after insult resulted in similar effects as those seen in rats treated with these Abs 24 h before Thy1 administration. Nevertheless, injection of AS101 starting 24 h after insult only mildly decreased STAT3 phosphorylation compared with the vast decrease seen when the compound was injected on day Ϫ1 (Figs. 8B and 9).
AS101 Affects STAT3-regulated Genes via Inhibition of IL-10 -Cyclin D1, the survival factors Bcl-2 and Bcl-X1, and the cyclin kinase inhibitor p27 kip1 have been previously been reported to be regulated by STAT3. We first aimed to ascertain whether this is also true for glomerular mesangial cells. Fig.  10A shows that mesangial cells transfected with antisense STAT3 oligonucleotides expressed decreased levels of both cyclin D1 and Bcl-2 but increased levels of p27kip1, implying a role for STAT3 in the regulation of these proteins in mesangial cells. Treatment of control mesangial cells transfected with control oligonucleotides with AS101 or anti-IL-10-neutralizing antibodies for 24 h resulted in similar alterations, whereas both treatments did not further decrease significantly the expression of these proteins in STAT3-AS-transfected cells. Mesangial cell proliferation requires a decrease in p27 kip1 levels, and the inhibitory threshold to growth factor-induced proliferation is determined by this CDK inhibitor. Fig. 10A shows that treatment of mesangial cells with either AS101 or anti-IL-10neutralizing antibodies increased the level of p27 kip1 only in control but not in STAT3-AS-transfected cells.
The effect of AS101 and anti-IL-10 treatments on these STAT3-regulated proteins was then examined in vivo. High expression of the survival proteins Bcl-2 and Bcl-X1 and of cyclin D1 was seen in glomerular lysates of Thy1-treated rats on day 6. Treatment with anti-IL-10 antibodies either before or after Thy1 administration greatly diminished expression of these proteins. AS101 given 24 h before insult, although somewhat less effective than ␣IL-10 treatment, significantly decreased the expression of all three regulatory proteins, whereas its administration after Thy1 was much less efficient. Fig. 10B shows low levels of glomerular p27kip1 in control Thy1-treated rats, which promptly increased in both ␣IL-10 and AS101treated rats on day 6. Again, treatment with AS101 after insult was less effective than its administration before Thy1. These data suggest a possible role of STAT3 in the regulation of cell cycle and survival of regulatory proteins by AS101 in mesangial cells via inhibition of IL-10.

DISCUSSION
In this study, we provide evidence that IL-10 is an important mesangial autocrine growth factor that plays an essential role in the pathogenesis of rat mesangioproliferative glomerulonephritis (GN). Its suppression by either anti-IL-10-neutralizing antibodies or by the tellurium anti-IL-10 compound, AS101, substantially ameliorated the disease as reflected by decreased glomerular mesangial expansion and reduced proteinuria. Furthermore, we have demonstrated that STAT3 is a key signaling molecule in IL-10-mediated MC proliferation in vitro and in vivo and its deactivation by AS101 is induced via inhibition of IL-10. We show, as was reported previously (25), that in Thy1 GN STAT3 is phosphorylated in glomerular cell nuclei, predominantly mesangial cells, and that this positively correlates with MC activation and proliferation. More importantly, interference with the IL-10-specific pathway extensively inhibited the phosphorylation of STAT3 in glomeruli in parallel with inhibition of glomerular MC proliferation.
Numerous cellular functions, such as growth, differentiation, and cell death, are regulated by cytokines. It is well established that the JAK/STAT signaling pathway plays a pivotal role in signal transduction via cytokine receptors (26). To date, seven members of the STAT family of proteins have been identified in mammals and each STAT protein has been implicated in in-tracellular signaling elicited by distinct cytokines (27). It has been shown that phosphotyrosine-based motifs residing in receptor subunits determine which particular STAT(s) is activated by a specific cytokine (28).
The binding of IL-10 to its cell surface receptors in a variety of cells activates the JAK/STAT signal transduction pathway (8). Following the ligand-receptor interaction, JAK1 (associated with IL-10R1) and Tyk2 (associated with IL-10R2), members of the receptor-associated JAK family, are phosphorylated (8). A family of latent cytoplasmic transcription factors, STATs, are then activated. Among the seven mammalian family mem- bers, STAT1, STAT3, and in some cells STAT5 are activated by . Further studies proved that STAT3 activation is essential for all of the known IL-10-mediated immune responses, although the activation of STAT3 alone is not sufficient for IL-10-mediated anti-inflammatory response (29). We show in this study for the first time that STAT3 activation is also essential for IL-10-mediated mesangial cell proliferation.
IL-10 has been previously demonstrated as a growth factor for rat mesangial cells in vitro (30). Furthermore, administration of IL-10 to normal rats induced the proliferation of glomerular MC in vivo (30). In addition, IL-10 gene and protein expression has also been observed within glomeruli from biopsies obtained from patients with IgA nephropathy (31), suggesting a role in human-mesangioproliferative glomerulonephritis. However, our study is the first to show the role of IL-10 in the pathogenesis of rat Thy1 mesangioproliferative GN. Our results contradict a recent study of Kitching et al. (32) who reported that IL-10 inhibits mesangial proliferative GN. However, in that study, no decrease in ␣-smooth muscle actin expression in glomeruli was noted. Rather, a decrease in infiltrating macrophages and in total glomerular cells was found. Alternatively, this apparent contradiction might be explained by differences in IL-10-mediated outcomes because of concentration-dependent effects and is also recognized in experimental tumor models. Although numerous studies describe the beneficial effects of neutralizing IL-10 on tumor regression, some reports describe the anti-tumoral effects of IL-10. However, they treat mice systemically with IL-10 at serum concentrations of around 500 -1000 ng/ml (33). These cytokine levels are not comparable with the amount of IL-10 secreted by tumors or tumor cell lines (0.04 -5 ng/ml) (34) and thus may be expected to have different effects. Cytokines may have opposite effects depending on their local concentration, and similar duality of effects has already been reported for other cytokines like tumor necrosis factor-␣, granulocyte-macrophage colonystimulating factor, or transforming growth factor-␤ (35)(36)(37).
Our study reveals that neutralization of IL-10 by neutralizing antibodies starting either 24 h before or 24 h after Thy1 administration greatly ameliorated the disease as expressed by both decreased MC expansion and proteinuria. Nevertheless, the efficiency of the anti-IL-10 compound AS101 was comparable to that of anti-IL-10-neutralizing antibodies only if administered starting 24 h before insult while its administration 24 h after Thy1 only partially decreased proteinuria. This may be explained by the immediate neutralization of IL-10 by neutralizing antibodies while prolonged time is needed to obtain a pronounced decrease in IL-10 following the administration of AS101. Although neutralization of IL-10 prevents the activity of IL-10, we show that it also affects the abundance of this cytokine. It probably suggests that this treatment results in the decrease of glomerular MC number for which IL-10 serves as a growth factor. Consequently, less IL-10 is produced. Experimental animal models such as anti-Thy1.1 nephritis exhibit marked proliferation of MCs leading to transient MC hyperplasia and matrix accumulation (2). If no repetitive insult to the mesangium is added in these animals, the disease shows spontaneous repair of the glomerular tufts within 2 weeks. This may explain the partial efficiency of AS101 when administered 24 h after the insult because the peak of mesangial proliferation occurs at 6 -7 days, whereas 48 h are needed for AS101 to significantly inhibit glomerular IL-10. In subsequent experiments, we have indeed seen that treatment of rats with anti-IL-10-neutralizing antibodies 48 h after Thy1 did not affect considerably glomerular pathology (data not shown), implying that amelioration of Thy1 GN requires IL-10 neutralization at the onset of disease. The administration of AS101 after the onset of glomerular injury may be found more effective in chronic glomerular progressive diseases. An apparent discrepancy in AS101 effects when administered after Thy1 resulting in partial albeit significant decreased proteinuria with no parallel improvement in glomerular pathology by light microscopy was found in this study. This apparent inconsistency may be attributed to subtle reparative histological changes because of the AS101 treatment that were not detected by light microscopy but attenuated the rate of proteinuria. Indeed, reparative changes appear early after the insult in this model of GN, which can be detected by electron microscopy only (38). It is conceivable that the treatment with AS101 24 h after Thy1 led to amelioration in sieving properties of the glomeruli such as improved charge selectivity. These changes may lead to reduction in proteinuria with no apparent change in either light or electron microscopy (39).
Inhibition of phosphorylated STAT3 expression in glomerular mesangial cells either in vitro or in vivo by both AS101 and anti-IL-10-neutralizing antibodies resulted in decreased expression of the major survival proteins Bcl-2 and Bcl-x. The in vitro data point to their STAT3-dependent inhibition by both treatments. Indeed, both survival factors have been previously reported to be regulated by STAT3 in a variety of cells (40).
It is intriguing to note that neutralization of IL-10 completely inhibited glomerular mesangial cell expansion despite the existence of many other potent growth factors known to FIG. 10. The effect of AS101 on STAT3-regulated genes is associated with inhibition of IL-10. MCs transfected with either control or STAT3 antisense oligonucleotides were cultured with either AS101 (0.5 g/ml) or ␣IL-10-neutralizing antibodies (10 ng/ml) for 24 h. Total cell lysates were subjected to immunoblotting with antibodies to cyclin D1, Bcl-2, or p27 kip1 (A). Glomeruli were isolated from kidneys collected from rats treated by different protocols on day 6 after Thy1 administration. Total cell lysates were subjected to immunoblotting with antibodies to cyclin D1, Bcl-2, or p27 kip1 (B). The results show one representative experiment of three performed. stimulate mesangial cell proliferation in Thy1 GN. Furthermore, disruption of either one of the other growth factor-mediated pathways also promptly inhibited the pathological manifestations of this disease. Even more appealing is the fact that at least some of these growth factors share overlapping modes of resolution of cell proliferation. Growth factor-induced mesangial cell proliferation is associated with decreased levels of p27 kip1 , and the onset of immune-mediated mesangial cell proliferation in experimental glomerulonephritis also coincides with a decrease in p27 kip1 levels (4). We show that inhibition of IL-10 results in the increase of p27 kip1 expression by glomerular cells. This is in line with IL-10-induced B cell proliferation, which has been also reported to be mediated by decreased p27 kip1 (41). Therefore, one might expect that lowering p27 kip1 levels via neutralization of any one of the mesangial growth factors will result in amelioration of the disease. Nevertheless, we have previously shown (6) that the decrease in p27 kip1 by transfection of anti-sense oligonucleotides, at least in vitro, is necessary albeit not sufficient for mesangial cell proliferation. However, once decreased, growth factor-mediated cell proliferation is induced more efficiently. Thus, besides lowering p27 kip1 , growth factors probably contribute further elements needed for mesangial cell proliferation. It may be conceivable that these growth factors act in concert and that despite their apparent redundancy they all must be present for supporting maximal mesangial proliferation. Such an interdependence between cytokines has been described previously in various physiologic states. Alternatively, growth factors may modulate the expression of each other. For example the mesangial mitogenic factor EGF induces the expression of PDGF, whereas blocking PDGF with anti-PDGF antibody abrogates the mitogenic effect of EGF-induced MC proliferation (42). Similarly, Gas6 induces mesangial cell proliferation via activation of STAT3 (25) and STAT3 was recently shown to activate the IL-10 promoter in macrophages (43).
Apart from the known ability of AS101 to inhibit IL-10, the beneficial pre-clinical effects of the compound have been attributed to this property. More importantly, AS101 was previously shown to delay the onset of autoimmune manifestations in a murine model of lupus erythematosus, reduce the level of immune complex deposition in the glomeruli, reduce proteinuria, prevent glomerular hypercellularity and mesangial expansion, and reduce the mean glomerular volume of treated mice (19). Furthermore, in a murine model of septic peritonitis, AS101 was recently shown to prevent kidney damage of septic mice (20). The above-mentioned effects of AS101 were attributed to the decreased levels of IL-10 in AS101-treated mice.
MC proliferation is a characteristic feature of many forms of glomerular diseases including IgA nephropathy, lupus nephritis, and mesangial and membrano-proliferative glomerulonephritis and is closely linked to matrix expansion (44). Because the proliferation of mesangial cells is essential for the pathogenesis and progression of glomerular diseases, several studies have attempted to suppress mesangial cell proliferation by inhibiting specific mitogens. However, interventions targeting these inhibitors to the site of proliferation are difficult and have been clinically used in only restricted situations.
We suggest that the use of the non-toxic compound AS101, currently undergoing phase II clinical trials, has potential in the clinical treatment of mesangioproliferative GN provided that it will be found effective in models of chronic progressive glomerular diseases when administered after injury. In addition, the involvement of STAT3 in mesangial IL-10 signaling pathways makes it an attractive target for interventional therapy in kidney diseases induced by mesangial proliferation.