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Centre for Inflammatory Diseases, Monash University Department of Medicine, Clayton, Victoria, Australia.
Correspondence to Dr. Peter G. Tipping, Centre for Inflammatory Diseases, Monash University Department of Medicine, Level 5, Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. Phone: +61 3 9594 5547; Fax: +61 3 9594 4279; E-mail: peter.tipping{at}med.monash.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production was abolished by high-dose treatment and reduced by the lower dose
(3.8 ± 3.8 pg/104 glomeruli per 72 h; control treatment: 249
± 23 pg/104 glomeruli per 72 h). These studies demonstrate
that IL-10 directly attenuates glomerular macrophage recruitment, activation,
and proliferation in vivo and can significantly attenuate
macrophage-mediated GN independent of any effects on T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interleukin-10 (IL-10) is an immunomodulatory cytokine with the potential to attenuate proliferative GN. It is capable of modifying nephritogenic immune responses at several levels. Macrophages are important effectors of Th1-directed immune responses. Administration of IL-10 attenuates T cell-induced macrophage-mediated crescentic GN in mice (9). However, in this model Th1 subset-directed nephritogenic immune responses were significantly attenuated, leaving undetermined what role, if any, IL-10 plays in deactivating glomerular macrophages independent of T cell direction.
In addition to its ability to attenuate active Th1 immune responses by
modulating the T cell response, IL-10 has been demonstrated to directly
inhibit macrophage activation and effector functions in vitro
(reviewed in reference (10).
The direct effects of IL-10 on macrophages in vivo are less well
characterized. IL-10 has been reported to decrease lipopolysaccharide (LPS)
and interferon-
or IL-1 -stimulated expression of tissue factor
(11,12).
IL-10 decreases the production of a number of proinflammatory cytokines,
including tumor necrosis factor- (TNF-), IL-1ß, and IL-6
(13,
14,
15), through the inhibition of
nuclear factor-
B in activated monocytes/macrophages
(16). The production of the
colony-stimulating factors M-CSF
(17), G-CSF, and GM-CSF
(18) and the stimulatory
effects of migration inhibitory factor on macrophages
(19) are inhibited by IL-10.
The effects of IL-10 on reactive oxygen and reactive nitrogen intermediate
release are more complex. Higher doses of IL-10 are required to inhibit
H2O2 and nitric oxide (NO) production than, for example,
to inhibit TNF- and IL-1ß production
(10,14).
Others have found that IL-10 enhances NO production
(20,21).
IL-10 also inhibits constitutive and cytokine-stimulated MHC class II
expression (22). In
vitro studies suggest that IL-10 has variable effects on adhesion
molecules (23,
24,
25) and chemokines
(26,27).
IL-10 has shown promising effects in human inflammatory diseases in which
macrophages are prominent, including inflammatory bowel disease
(28) and psoriasis
(29). It has the potential to
play a role in forms of GN, in which macrophage influx is prominent.
These in vitro inhibitory effects of IL-10 on macrophages suggest the possibility that similar direct in vivo effects on the macrophage functions may contribute to the capacity of IL-10 to attenuate crescentic GN. Models of experimental GN induced by active immune responses cannot dissect the direct effects of IL-10 on macrophages from its effects on T cell subset development and antibody production. We therefore used a model of macrophage-mediated proliferative GN induced by passive antibody transfer to assess the direct effects of IL-10 on glomerular macrophage recruitment, activation, and mediation of glomerular injury. This model is characterized by the influx of macrophages attracted by passively administered autologous antibody (4,30). CD4+ T cells are not present in the glomerular lesion, and unlike active autologous GN, CD4+ T cell depletion does not affect glomerular injury (30).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Passive autologous anti-GBM GN was induced in male Wistar Kyoto rats (150 to 200 g body wt) by injecting a subnephritogenic dose (5 mg/100 g body wt) of sheep anti-rat GBM globulin, which does not induce proteinuria in normal rats (30). This was followed 16 h later by intravenous injection of a single dose (10 mg/100 g) of rat anti-sheep globulin antibody. Injury was assessed 24 h after the administration of autologous antibody. This model has been demonstrated previously to be CD4+ T cell-independent (30). In the current study, the contribution of T cells to glomerular injury was assessed by treating six rats with 5 mg of monoclonal anti-rat CD5 antibody (OX19), given as a single intravenous dose 4 h before administration of sheep anti-rat GBM globulin. Injury was assessed after inducing GN as described above.
Rats were treated with recombinant murine IL-10 (specific activity 6.3 x 107 U/mg; Schering-Plough Research Institute, Kenilworth, NJ), which has been demonstrated previously to be active in a rat model of immune lung injury (31). IL-10 was diluted in sterile saline, and an equivalent volume of saline alone was used for control treatment. The timing of IL-10 or saline injections was as follows: the first dose was given 6 h before administration of anti-GBM globulin, the second 1 h before, and the third 1 h before injection of rat anti-sheep globulin antibody. The following groups were studied: (1) normal rats without GN or treatment (n = 8); (2) control (saline)-treated rats with GN (n = 8); (3) IL-10 low dose-treated rats with GN (3 doses of 25 µg/100 g) (n = 8); (4) IL-10 high dose-treated rats with GN (3 doses of 50 µg/100 g) (n = 8).
Measurement of Glomerular Deposition of Sheep Anti-GBM and Rat
Anti-Sheep Globulin Antibodies
The effect of IL-10 treatment on the glomerular deposition of sheep
anti-GBM and rat anti-sheep globulin antibodies was measured using the end
point titer for detection by immunofluorescence. Cryostat cut (4 µm) tissue
sections were cut from snap-frozen kidney and stained by direct
immunofluorescence with serial dilutions of FITC-conjugated rabbit anti-sheep
Ig (Cappel, Durham, NC) or FITC-conjugated rabbit anti-rat Ig (Silenus,
Hawthorn, Victoria, Australia). Normal rat renal tissue was used to assess
background fluorescence. Positive linear staining for Ig was assessed using a
blinded protocol in high-dose IL-10 (n = 6) and control-treated rats
(n = 6). The highest dilution of detecting antibody at which staining
above background levels could be detected was taken as the end point titer for
each animal.
Measurement of Proteinuria
Urine was collected during the final 24 h of the experiment from all rats
(eight in each group). Collections began immediately after injection of
autologous antibody. Urinary protein concentration was measured by the
Bradford assay (32), and 24-h
protein excretion was calculated by multiplying the urinary protein excretion
by the 24-h urine volume.
Histologic Analysis and Immunostaining
Analyses were performed on all animals (eight rats from each group). Renal
tissues were fixed in Bouin's fixative and embedded in paraffin. Tissue
sections (2 µm) were stained with periodic acid-Schiff reagent, and cell
nuclei were counted in a minimum of 20 equatorially sectioned glomeruli per
animal. Neutrophils were identified by the typical nuclear morphology. A
three-layer immunohistochemistry technique was used to assess macrophages, MHC
II, IL-1ß, monocyte chemoattractant protein-1 (MCP-1), P-selectin, and
proliferating cell nuclear antigen (PCNA) on paraffin or periodatelysine
paraformaldehyde fixed sections. The primary antibodies were: mouse anti-rat
macrophage (ED1; American Type Culture Collection [ATCC], Manassas, VA), mouse
anti-rat MHC class II (OX-6; ATCC), mouse anti-rat IL-1ß (Silk 6;
Serotec, Oxford, United Kingdom), rabbit anti-rat MCP-1 (a gift from Dr. R
Stahl, Department of Medicine, University of Hamburg, Germany), rabbit
anti-human P-selectin (a gift from Dr. Berndt, Baker Medical Research
Institute, Praharn, Victoria, Australia), and mouse anti-human PCNA (Dako,
Glostrup, Denmark). The anti-P selectin antibody has been previously
demonstrated to detect and functionally inhibit rat P-selectin
(33). The anti-MCP-1 antibody
has also been demonstrated to detect rat MCP-1
(34). An irrelevant mouse
monoclonal antibody or normal rabbit Ig was used as controls for the primary
antibodies.
Sections were treated by a microwave technique (35) before staining. Macrophage (ED1) staining was detected using mouse antibody to alkaline phosphatase and alkaline phosphatase complex (mouse APAAP; Dako) and Fast Blue BB Salt (Sigma Chemical Co., St. Louis, MO) as the substrate. Before staining the second antigen, sections were microwave-treated again. This protocol has previously been demonstrated to prevent cross-reactivity of subsequent antibodies used for detecting the primary antigen (35). MHC II staining was detected using mouse antibody to horseradish peroxidase and horse-radish peroxidase complex (mouse PAP; Dako). MCP-1 and P-selectin were detected using rabbit antibody to horseradish peroxidase and horseradish peroxidase complex (rabbit PAP; Dako). IL-1ß staining was enhanced using the Tyramide Signal Amplification kit (Dupont, Boston, MA), which uses 3,3-diaminobenzidine (Sigma) as the substrate.
For assessment of ED1, MHC II, IL-1ß, and PCNA, a minimum of 20 equatorially sectioned glomeruli per animal were assessed and the results were expressed as cells per glomerular cross section (c/gcs). Expression of MCP-1 and P-selectin in glomeruli was graded semi-quantitatively. No staining in any glomerulus was graded as 0. Positive staining present only in some glomeruli was graded ±. Animals with staining in all glomeruli were graded 1+ to 3+ according to the intensity of staining.
Measurement of Nitrite and TNF- Production by Glomeruli
Glomeruli were isolated by graded sieving from one kidney of six rats from
each group with GN. Glomeruli (1 x 104/ml) with a purity of
>95% were cultured in modified Eagle's medium with 10% fetal calf serum and
1% penicillin/streptomycin for 72 h (37°C, 5% CO2). NO was
assessed by accumulation of nitrite in glomerular culture supernatants,
measured using the Greiss reaction. Greiss reagent was prepared by mixing 1%
sulfanilamide (p-aminobenzene-sulfonamide; Sigma) in 1 M hydrochloric acid
(Ajax Chemicals, Sydney, Australia) with 0.15% naphthyldiamine
(N-(1-napthyl) ethyl-diamine dihydrochloride; Sigma) at a ratio of
1:1. Equal volumes (100 µl) of Greiss reagent and supernatant were mixed in
a 96-well plate, and the absorbance was read at 543 nm. Nitrite concentration
was determined using sodium nitrite (Ajax Chemicals) as a standard, and the
results were expressed as nmol nitrite/104 glomeruli per 72 h.
The production of TNF- by glomeruli was assessed by enzyme-linked
immunosorbent assay performed on supernatants from glomerular cultures (100
µl), via a commercial rat TNF- assay (Factor test X; Genzyme,
Cambridge, MA), and results were expressed as pg TNF-/104
glomeruli per 72 h. This assay has a sensitivity of 10 pg/ml (equivalent to 10
pg/104 glomeruli per 72 h).
Statistical Analyses
Results are expressed as the mean ± SEM. The statistical
significance of differences between groups was performed by ANOVA followed by
the Tukey multiple comparison test for paired comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects of IL-10 Treatment on Proteinuria and Proliferative GN
IL-10 treatment significantly attenuated glomerular injury in a
dose-dependent manner. Proteinuria was reduced to 30 ± 2 mg/24 h in the
low-dose IL-10-treated group and 16 ± 1 mg/24 h in the high-dose
IL-10-treated group (both P < 0.001 compared with control
treatment) (Figure 1A). IL-10
treatment also reduced the histologic severity of proliferative GN. Total
glomerular cell numbers were reduced in a dose-dependent manner
(Figure 1B) such that there was
no significant difference between the high-dose IL-10 treatment group and
normal rats without GN. Glomerular macrophage accumulation was significantly
reduced in IL-10-treated groups (low dose: 5.5 ± 0.2 c/gcs; high dose:
1.8 ± 0.1 c/gcs; both P < 0.001 compared to control
treatment) (Figure 1C).
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Effects of IL-10 Treatment on Glomerular Deposition of Sheep Anti-GBM
and Rat Anti-Sheep Antibodies
IL-10 treatment at the highest dose did not affect the glomerular
deposition of sheep anti-rat GBM globulin (end point titer for detection:
treated 1 in 5000; untreated 1 in 5000) or rat Ig (end point titer for
detection: treated 1 in 2000; untreated 1 in 2000).
Effects of IL-10 Treatment on P-Selectin and MCP-1 in Glomeruli
In control-treated rats with GN, expression of both P-selectin and MCP-1
was upregulated and observed predominantly on the glomerular endothelium
(Figure 2). IL-10 treatment
markedly attenuated the expression of P-selectin and abolished MCP-1
expression (Table 1),
demonstrating anti-inflammatory effects of IL-10 on both adhesion molecule and
chemokine expression in GN.
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Effects of IL-10 Treatment on Macrophage Activation and Effector
Functions
Effects of IL-10 on MHC Class II Expression. Dual expression of MHC
class II and ED1 was observed on 9.6 ± 0.4 c/gcs in control-treated
rats with GN (Figure 3A),
representing 78 ± 3% of ED1+ cells in glomeruli. Expression
of MHC II by ED1+ cells in glomeruli was reduced by IL-10 treatment
(low dose: 0.8 ± 0.1 c/gcs; high dose: 0.2 ± 0.03 c/gcs; both
P < 0.001 compared to control treatment)
(Figure 3, B and C). In
low-dose IL-10-treated rats, 15 ± 1% of macrophages expressed MHC II
and with the higher dose treatment, 5 ± 1% of glomerular macrophages
expressed MHC II, suggesting that in addition to inhibiting recruitment, IL-10
inhibits expression of this activation marker on macrophages within the
glomerulus.
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Effects of IL-10 on Glomerular TNF- Production and Macrophage
IL-1ß Expression. Isolated glomeruli from control rats with GN
produced TNF- (249 ± 23 pg/104 glomeruli per 72 h)
ex vivo (Figure 4A).
Low-dose IL-10 treatment markedly attenuated TNF- production, with
detectable levels in only one of six animals (23 pg/104 glomeruli
per 72 h). TNF- production was undetectable in glomerular supernatants
from rats treated with high-dose IL-10. Dual immunohistochemical staining
demonstrated IL-1ß expression on 3.8 ± 0.1 ED1+ c/gcs
in control-treated rats with GN, representing 32 ± 2% of
ED1+ macrophages (Figure
3D). Both low-dose and high-dose IL-10 treatment reduced the
numbers (Figure 3, E and F, and
Figure 4B) and percentage of
IL-1ß-positive macrophages in glomeruli (low dose: 4 ± 0.5%
ED1+ cells positive for IL-1ß; high dose: 3 ± 1%
positive; both P < 0.001 compared to control-treated rats).
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Effects on Glomerular Nitrite Production. Nitrite, a stable degradation product of NO, was measured in supernatants of cultured glomeruli to provide an index of NO production. Nitrite production was reduced in a dose-dependent manner by IL-10 treatment (low dose: 28 ± 5 nmol/104 glomeruli per 72 h; high dose: 2.3 ± 2.3 nmol/104 glomeruli per 72 h; both P < 0.001 compared to control-treated rats: 82 ± 11 nmol/104 glomeruli per 72 h) (Figure 4C).
Effects of IL-10 on Macrophage Proliferation and Glomerular Cell
Proliferation
The effects of IL-10 treatment on cellular proliferation in glomeruli was
assessed by immunostaining for PCNA. Control-treated rats had 5.5 ± 0.3
PCNA+ c/gcs. Dual immunostaining revealed that 3.3 ± 0.4 of
these PCNA+ cells were ED1+ macrophages
(Figure 3G). PCNA was expressed
by 28 ± 2% glomerular macrophages in control-treated rats. Treatment
with IL-10 resulted in a dose-dependent reduction in PCNA+ cells
within glomeruli. The numbers of both proliferating macrophages
(PCNA+ ED1+ cells) and proliferating intrinsic
glomerular cells (PCNA+ ED1- cells) were reduced
(Figure 3, H and I). The
percentage of macrophages expressing PCNA was also reduced by IL-10 treatment
(low dose: 7 ± 1%; high dose: 6 ± 3%)
(Figure 5), indicating that
IL-10 reduces macrophage proliferation in glomeruli, in addition to its
effects on macrophage recruitment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor-dependent macrophage interaction
(3,6,30).
Treatment with recombinant murine IL-10 inhibited macrophage recruitment,
activation, and proliferation in this model in a dose-dependent manner. This
inhibition of macrophage recruitment and function resulted in a significant
reduction in proteinuria. IL-10 has previously been demonstrated to reduce
immune glomerular injury and macrophage recruitment in CD4+ T
cell-dependent experimental GN
(9). The results of the current
study demonstrate that effects on macrophage recruitment and activation,
independent of effects Th1 immune responses, contribute to the capacity of
IL-10 to protect against immune renal injury. Because IL-10 therapy is currently being used as therapy for human inflammatory diseases, knowledge of its in vivo biology is particularly relevant. (28,29). Previous studies have demonstrated T cell-dependent effects of IL-10 on other cellular inflammatory mediators in vivo (36, 37, 38, 39). Direct effects on neutrophils have been suggested by studies of acute IgG immune complex-induced lung injury in rats (40). In this model, in which neutrophils rather than macrophages are the predominant inflammatory cell, treatment with IL-10 has been shown to provide protection against lung injury. Suppression of resistance to Listeria monocytogenes infection in severe combined immunodeficient mice suggests that IL-10 can also directly affect natural killer cell function (41).
In vitro, IL-10 has been demonstrated to directly inhibit
macrophage activation and effector functions, including MHC class II
expression and TNF- and IL-1ß production
(10). Previous studies provide
some evidence for direct effects of IL-10 on macrophage function. In
experimental endotoxic shock and in human endotoxemia, IL-10 inhibits
TNF- production
(42,43)
and activation of the coagulation system
(44), suggesting direct
effects on macrophage activation in vivo, although IL-10 also
inhibited neutrophil responses in human endotoxemia
(43).
The current studies provide in vivo evidence for direct effects of
IL-10 on a variety of macrophage activation markers and effector molecules.
Activation of macrophages within glomeruli was assessed by the expression of
MHC II and IL-1ß. IL-10 administration decreased the proportion of
macrophages within glomerulus expressing MHC II. IL-1 and TNF- have
been shown to be relevant effector molecules in GN
(45,
46,
47). IL-10 treatment produced
dose-dependent decreases in both IL-1 and TNF- production in
vivo. Effects of IL-10 on production of TNF- by cultured glomeruli
are likely to be attributable to effects on macrophages, as these cells have
previously been demonstrated to be the major cell type responsible for
glomerular TNF- production in macrophage-dependent experimental GN
(47). However, effects on
intrinsic glomerular cells cannot be discounted, since IL-10 has been
demonstrated to attenuate TNF production by LPS-stimulated mesangial cells
in vitro (48).
In vivo, IL-10 treatment also reduced nitrite production by
isolated cultured glomeruli. Nitrite is a sensitive index of NO production.
Although there was a significant reduction in glomerular nitrite production in
rats treated with the lower dose of IL-10, nitrite production fell to
unmeasurable levels in all but one of the rats given the higher dose,
suggesting that higher doses of IL-10 are required to abolish NO than
TNF- production. This finding is consistent with the study of Bogdan
et al. (14), which
demonstrated that higher doses of IL-10 are needed to inhibit NO production
than to inhibit cytokines. Intrinsic glomerular cells may also contribute to
nitrite production in cultured glomeruli, although in vitro evidence
suggests that IL-10 does not attenuate NO production by LPS-stimulated
mesangial cells in vitro
(48).
Studies of the glomerular expression of adhesion molecules and chemokines after IL-10 treatment provide some insight into potential mechanisms of reduced macrophage recruitment. Expression of both P-selectin and MCP-1 was downregulated in a dose-dependent manner. Both of these molecules have been reported to play a role in inflammatory leukocyte recruitment in experimental GN (7,8).
Macrophages have been shown to proliferate within glomeruli in GN, and it has been suggested that the proliferative index is related to the severity of glomerular injury (49,50). IL-10 treatment reduced the numbers of proliferating ED1+ macrophages and the percentage of macrophages expressing PCNA. Proliferation of PCNA+/ED1- intrinsic glomerular cells was also reduced by IL-10 treatment. It is unknown whether the effects on intrinsic glomerular cell proliferation were a consequence of the inhibitory effects of IL-10 on macrophages or a direct effect of IL-10 on these cells.
In summary, this study shows that IL-10 attenuates injury in a macrophage-dependent, T cell-independent model of GN via effects on macrophage recruitment, activation, and function. In addition to its inhibitory effects on the development of the Th1 subset nephritogenic immune responses, IL-10 exerts its beneficial effects in macrophage-dependent GN via its capacity to directly inhibit macrophage effector functions.
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Acknowledgments |
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enzyme-linked
immunosorbent assay. |
References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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