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*
Nephrologisches Zentrum, Medizinische Poliklinik,
Ludwig-Maximilians-University, Munich, Germany.
Department of Animal Physiology, Ludwig-Maximilians-University, Munich,
Germany.
Department of Nephrology and Rheumatology, University of
Göttingen,
Göttingen, Germany.
Department of Cellular and Molecular Pathology, German Cancer Research
Center, Heidelberg, Germany.
Correspondence to Professor Dr. Detlef Schlöndorff, Medizinische Poliklinik, Ludwig-Maximilians-University, Pettenkoferstrasse 8a, 80336 Munich, Germany. Phone: ++49-89-51603500; Fax: ++49-89-51604439; E-mail: sdorff{at}pk-i.med.uni-muenchen.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines, an expanding family of small chemotactic cytokines, play a
major role in attracting circulating leukocytes into inflamed renal tissue in
a variety of kidney diseases
(7,8).
According to the position of conserved cysteine residues in their primary
sequence, the chemokine superfamily is divided into four subgroups (C, CC,
CXC, and CX3C chemokines), which attract specific subsets of
leukocytes (9). Chemokine
expression secondary to stimulation with proinflammatory cytokines has been
reported in many types of intrinsic renal cells in vitro and in
vivo, including tubular epithelial, interstitial, endothelial, and
mesangial (8). The biologic
effect of chemokines on the target leukocytes is mediated through seven
transmembrane-spanning G-protein—linked receptors, which are specific
for individual chemokine subgroups. Within one subgroup, multiple chemokines
can bind to a single receptor, which leads to a certain degree of redundancy
(8,9).
The pivotal role for chemokines in the pathogenesis of renal inflammation has
been verified by blocking chemokine activity with neutralizing antibodies,
chemokine receptor antagonists, and targeted chemokine gene disruption in
various animal models
(8,10).
The CC chemokines monocyte chemoattractant protein-1 (MCP-1)/CCL2,
RANTES/CCL5, and macrophage inflammatory protein-1
(MIP-1)/CCL3
emerged as important mediators of renal leukocyte attraction and inflammation
by these neutralization studies
(11,12,13,14,15,16,17,18,19,20).
In two animal models of glomerulonephritis, administration of anti-MCP-1
antiserum or the CC chemokine receptor antagonist AOP-RANTES decreased not
only glomerular leukocyte infiltrates but also glomerular and interstitial
collagen deposition
(13,15,17).
Thus, CC chemokines and their receptors might also play a role in mediating
tubulointerstitial leukocyte recruitment, local inflammation, and subsequent
tubulointerstitial fibrosis.
However, the expression pattern and role of CC chemokines and particularly their specific receptors in tubulointerstitial inflammation and progressive fibrosis have not been investigated. We used unilateral ureteral obstruction (UUO) in C57B1/6 mice as a model, which is characterized by an infiltration of macrophages and T cells into the interstitium and concomitant renal fibrosis (6,21). In the present investigation, we demonstrate a progressively increasing tubulointerstitial expression of MCP-1 and RANTES during the development of obstructive nephropathy, which correlates with a simultaneous interstitial accumulation of macrophages and lymphocytes expressing the respective surface receptors CCR2 and CCR5 and a progressive interstitial fibrosis. Blockade of the CC chemokine receptor—mediated leukocyte influx by chemokine receptor antagonists therefore may offer a new therapeutic approach for progressive interstitial fibrosis in chronic renal disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under general ether anesthesia, unilateral ureteral ligation resulting in UUO was performed by ligating the left distal ureter with a 2/0 Mersilene suture through a low midline abdominal incision. Unobstructed contralateral kidneys served as controls. Groups of mice were killed at 2, 6, and 10 d after UUO by cervical dislocation under general anesthesia with inhaled ether.
Renal Morphology and Immunohistochemistry
From each mouse cranial kidney halves were used for histologic assessment.
Ligated and contralateral kidney tissue was fixed for 24 h at room temperature
in 4% neutral buffered formalin and then embedded in paraffin. For
quantitative analysis, 4-µm horizontal sections were cut. Every fifth of 15
subsequent sections, chosen by systematic uniformly random sampling, was used
for analysis (22). Slides were
stained with periodic acid-Schiff (PAS) reagent for routine histology and
morphometric analysis. For immunohistologic studies, sections were
deparaffinized, rehydrated, transferred into citrate buffer, and either
autoclaved or microwave treated. Sections were blocked with 3% peroxidase,
avidin, and biotin (Vector Blocking Kit; Vector Laboratories, Burlingame, CA)
for 20 min each. After slides were washed in phosphate-buffered saline, they
were incubated with the primary antibody for 1 h at room temperature. The
following monoclonal rat antibodies were used: anti-mCD45 against leukocytes
(Pharmingen, San Diego, CA; 1:200), anti-F4/80 against macrophages (Serotec,
Oxford, UK; 1:50), anti-mCD3 against human lymphocytes (Serotec; 1:100), and
anti-type IV collagen (Research Diagnostics, Flanders, NY; 1:100). Activated
fibroblasts were stained with a polyclonal antiserum (1:1000) against
recombinant murine fibroblast-specific protein 1 (FSP1) generated in a New
Zealand white rabbit as described elsewhere
(23). For mRANTES/CCL5, a
polyclonal rabbit anti-mouse antibody (PeproTech, Rocky Hill, NY; 1:50) was
used. For detection of mMCP-1/CCL2, a custom antipeptide antiserum against
murine MCP-1 was raised in rabbits as described previously
(24). Signals of all primary
antibodies were detected with a commercial mouse link and label kit following
the instructions of the supplier (SuperSensitive; BioGenex, San Ramon, CA).
3-amino-9-ethylcarbazole substrate was used for signal development. All
sections were counterstained with hemalaun. For quantitation of the
interstitial cell infiltrate, 20 cortical high-power fields (hpf, 400x)
per kidney were selected randomly by a blinded observer and the mean cell
count in each kidney was expressed as cells per hpf.
Morphometric Analysis of Tubular Damage, Interstitial Volume, and
Interstitial Type IV Collagen Deposition
Markers of tubular damage and interstitial fibrosis were assessed
morphometrically (25). In
brief, a grid containing 117 (13 x 9) sampling points was superimposed
on images of cortical hpf (400x) from sections stained with PAS reagent
or type IV collagen antibody. The number of grid points overlying the tubular
lumen (tubular dilation index), atrophic or necrotic tubular cells (index of
tubular cell damage), interstitial space (interstitial volume index), and
interstitial type IV collagen deposits (interstitial collagen IV index) was
counted and expressed as a percentage of all sampling points. For each kidney,
10 randomly selected, nonoverlapping fields were analyzed by a blinded
observer.
In Situ Hybridization
Single-stranded radiolabeled riboprobes for mMCP-1/CCL2, mRANTES/CCL5,
mCCR2, and mCCR5 were prepared by in vitro transcription of subcloned
cDNA fragments. The 161-bp MCP-1 probe corresponds to nucleotides 299 to 459
(GenBank accession number, J04467) and the 321-bp mRANTES probe corresponds to
nucleotides 124 to 444 (GenBank accession number, S37648) of the respective
cDNA sequence. For mCCR2 and mCCR5 probes, cDNA fragments in the untranslated
5' and 3' end, respectively, with low sequence homology were
subcloned. The 153-bp mCCR2 probe represents nucleotides 1736 to 1888 (GenBank
accession number, U47035), and the 220-bp mCCR5 probe represents nucleotides
1384 to 1604 (GenBank accession number, D83648). Antisense and sense RNA
transcripts were labeled with 35S-UTP (1250 Ci/mmol; NEN,
Cologne, Germany) to a specific activity of 8 x 108 cpm/µg
and served as hybridization probe and control, respectively. In situ
hybridization was performed as described previously following modified
standard protocols (24).
Briefly, 4-µm paraffin sections were digested with 5 µg/ml proteinase K
and acetylated. Prehybridization was performed in 50% formamide, 0.3 M NaCl,
10 mM sodium phosphate buffer, 10 mM Tris-HCl, 5 mM
ethylenediaminetetraacetate (EDTA; pH 7.4), 10% dextransulfate, 25 mM DTT, 1
x Denhardt's, and 1.25 mg/ml tRNA at 52°C for 2 h. Sections were
hybridized at 52°C overnight with 40 µl of prehybridization solution
containing labeled RNA probe with an activity of 0.8 x 105
cpm. After hybridization, four washes in 4 x SSC were followed by a
washing procedure with 50% formamide, 1 x SSC at 50°C for 60 min.
Sections then were incubated in a mixture of RNase A (40 µg/ml) and RNase
T1 (50 units/ml) at 37°C for 30 min, followed by two high-stringency
washes in 0.1 x SSC (37°C, 15 min). Slides were dehydrated, dipped
in Ilford K2 nuclear research emulsion (Ilford, Mobberly, UK) diluted 1:1 with
distilled water at 42°C, and exposed for 2 to 4 wk at 4°C in a dry
chamber. After development, sections were counterstained with Harris
hematoxylin and eosin. In situ hybridization for chemokine receptors
and immunostaining for CD3+ cells was combined on some sections. After in
situ hybridization, immunohistochemistry was performed as described above
without autoclaving or microwave pretreatment. After developing the
3-amino-9-ethylcarbazole substrate, slides were dipped, exposed, developed,
and counterstained with hematoxylin.
RNA Preparation and Renal Chemokine and Chemokine Receptor
Expression
The lower half of each kidney was snap-frozen in liquid nitrogen and stored
at -80°C. Total RNA was prepared with the use of the method of Chomczynski
and Sacci (26). Chemokine and
chemokine receptor expression was analyzed with the use of ribonuclease
protection assays (RPA). RiboQuant multiprobe template sets for murine
chemokines (mCK-5) and murine CC chemokine receptors (mCR-5) were obtained
from Pharmingen. Twenty µg (chemokines) or 50 µg (receptors) of total
RNA were used for each determination. Transcription of antisense riboprobes
with 32P-UTP (3000 Ci/mmol; NEN), hybridization, and RNase
treatment was performed according to the manufacturer's instructions. Efficacy
of the RNase step was ensured by a yeast t-RNA sample in every assay as a
negative control. RNase protected hybridization products were separated on a
6% denaturing polyacrylamide gel. The intensity of the specific bands was
quantitated with the use of a Storm 840 PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) and a standard software program (Image-Quant; Molecular
Dynamics) and normalized to glyceraldehyde phosphate dehydrogenase gene
expression.
Isolation of Renal Cells
A preparation of isolated renal cells, including inflammatory leukocytes,
was obtained from obstructed kidneys and contralateral control kidneys 10 d
after UUO, following a method adapted from Cook et al.
(27). In a first enzyme step,
mechanically disaggregated tissue was incubated for 20 min in 5 ml of Hanks'
balanced salt solution (1 x HBSS) containing 1 mg/ml collagenase type I
and 0.1 mg/ml deoxyribonuclease type III (both from Sigma, Deisenhofen,
Germany) at 37°C. Trypsin was omitted to avoid degradation of cell surface
antigens, especially CD4 and CD8. After incubation in 5 ml of 2 mM EDTA, 1
x HBSS (without calcium and magnesium) for 20 min at 37°C, the
supernatant containing isolated cells was removed and kept on ice. In a second
enzyme step, the remaining pellet was incubated in 5 ml of 1 mg/ml collagenase
I in 1 x HBSS for 20 min at 37°C. The resulting supernatant
containing free cells was pooled with the first supernatant from the EDTA
incubation, washed twice in 1 x HBSS, and resuspended in 1 ml of the
same buffer. After staining with 0.4% trypan-blue, cells were counted in a
Neubauer chamber. Typically, 7 to 8 x 105 cells/ml could be
isolated from two pooled kidneys. Before kidneys were removed for cell
isolation, blood samples were taken from the anesthetized mice by retrobulbar
puncture, collected in 4 mM EDTA and stored on ice until labeling for flow
cytometry.
Flow Cytometry
Rat monoclonal antibodies to detect murine CCR2 and CCR5 expression on
isolated leukocytes were generated as described elsewhere
(28). Renal cell suspensions
and anticoagulated full blood samples were incubated with 5 µg/ml of the
monoclonal antibodies MC-21 or MC-68 for 60 min on ice. The antibody MC-21
binds specifically to murine CCR2, and the antibody MC-68 binds to murine
CCR5. As isotype control, samples also were stained with rat IgG2b
(Pharmingen). After 3 washing steps, cells were incubated for 60 min on ice
with a biotin-labeled anti-rat polyclonal antibody followed by
phycoerythrin-labeled streptavidin (both from Dako, Hamburg, Germany). For the
identification of leukocyte subsets, samples were finally incubated with a
combination of the following directly conjugated cell-specific antibodies:
CD11b fluorescein-isothiocyanate (clone M1/70), CD4 allophycocyanin, and CD8
cy-chrome (all from Pharmingen). After lysis of erythrocytes with
fluorescence-activated cell sorter (FACS)-lysing solution (Becton-Dickinson,
Franklin-Lakes, NJ), stained cells were analyzed on a flow cytometer
(FACS-calibur, Becton-Dickinson). Monocytes/macrophages were identified by
their light scatter properties and expression of CD11b; T lymphocytes were
identified by expression of CD4 and CD8. The cutoff to define chemokine
receptor—positive cells was set according to the staining with the
isotype control antibody. For CCR2 and CCR5 expression on macrophages, shifts
of the mean fluorescence compared with isotype control are given, as some
unspecific binding of the IgG2b antibody on activated renal macrophages
occurred. Approximately 100,000 gated events were collected in each
analysis.
Statistical Analyses
Values are expressed as mean ± SD. Statistical analysis was
performed with the use of the t test for unpaired data (RPA) or
paired data (morphometric analysis). Statistical significance was defined as
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Histopathologic Changes
Renal tubular damage after UUO increased progressively from days 2 to 10,
involving the cortex and the medulla. Tubular damage was characterized by
dilation with flattened epithelium, atrophy, and necrosis
(Figure 1, A and B;
Figure 2A;
Table 1). In parallel, a
progressive expansion of the interstitial space and an increased extracellular
matrix deposition occurred in the cortex
(Table 1), as revealed by PAS
staining and immunohistochemical detection of type IV collagen deposits
(Figure 1, E and F;
Figure 2A). In addition,
fibrotic changes were characterized by the progressive accumulation of
interstitial FSP1 + fibroblasts from day 2 (8.8 ± 1.1 versus
5.0 ± 0.8 cells/hpf in the contralateral kidneys) to day 10 (20.0
± 2.8 versus 4.7 ± 1.5 cells/hpf)
(Figure 1, C and D;
Figure 2A;
Table 1. Results are mean
± SD from five mice per time point). FSP1 recently was characterized as
a marker for activated fibroblasts contributing to tissue fibrogenesis. It is
a S100A4 protein expressed constitutively in the cytoplasm of resident tissue
fibroblasts (23). No gross
glomerular pathology was evident in mice with UUO at any time point.
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Accumulation of Interstitial Macrophages and Lymphocytes in
Obstructed Kidneys
Increasing numbers of infiltrating CD45+ leukocytes were identified in the
cortical interstitium at 2, 6, and 10 d after UUO by immunohistochemical
staining (Table 1). At 2 and 6
d, more F4/80+ macrophages than CD3+ lymphocytes could be detected in the
mononuclear cell infiltrate, whereas at 10 d after UUO approximately 60% of
infiltrating cells were lymphocytes (Figure
1, G through J; Figure
2B; Table 1.
Results are mean ± SD from five mice per time point). Maximal numbers
of interstitial macrophages were observed at day 6 (10.9 ± 1.5
versus 1.5 ± 0.4 cells/hpf in the control kidneys), with a
decline at day 10 (8.7 ± 1.4 versus 2.1 ± 0.4
cells/hpf). In contrast, the interstitial influx of lymphocytes increased
progressively from day 2 (0.3 ± 0.1 versus 0.1 ± 0.0
cells/hpf) to day 10 (12.4 ± 1.4 versus 0.6 ± 0.1
cells/hpf). In glomeruli of obstructed kidneys, no leukocyte infiltrates
(CD45+, F4/80+, CD3+ cells) could be detected.
Increased Chemokine mRNA Expression in Obstructed Kidneys
Expression of chemokines involved in the recruitment of macrophages and
lymphocytes to sites of interstitial inflammation was determined by RPA.
Whole-organ mRNA samples from obstructed and contralateral kidneys were
obtained from 4 mice at each time point. The mRNA levels of
lymphotactin/CXCL1, RANTES/CCL5, eotaxin/CCL11, MIP-1/CCL3,
MIP-1ß/CCL4, MIP-2, 
-interferon-inducible protein-10
(IP-10)/CXCL10, MCP-1/CCL2, and TCA-3/CCL1 were determined relative to the
internal standards L32 and glyceraldehyde phosphate dehydrogenase. At 2 d
after UUO, no significant increase of tested chemokine mRNA levels was evident
in obstructed kidneys compared with contralateral controls, with the exception
of a slightly elevated TCA-3 expression
(Figure 3). At day 6,
obstructed kidneys contained significantly more mRNA coding for MCP-1/CCL2,
RANTES/CCL5, MIP-2, and IP-10/CXCL10 relative to contralateral control kidneys
(Figure 3). There was also a
slight but significant increase of lymphotactin/XCL1, eotaxin/CCL11,
MIP-1/CCL3, and MIP-1ß/CCL4 mRNA expression. Ten d after UUO, a
further induction of MCP-1/CCL2 (60-fold compared with contralateral
controls), MIP-2 (55-fold), RANTES/CCL5 (21-fold), and IP-10/CXCL10 (11-fold)
mRNA could be detected (Figure
3). In addition, a further upregulation of lymphotactin/XCL1
(8.7-fold), MIP-1/CCL3 (7.8-fold), MIP-1ß/CCL4 (7.5-fold), and
eotaxin/CCL11 (4.7-fold) mRNA expression occurred. Increased TCA-3 mRNA levels
were present at all time points.
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Tubulointerstitial Localization of MCP-1 and RANTES mRNA and
Protein
To localize the sites of chemokine mRNA expression, we performed in
situ hybridization on paraffin-embedded sections with antisense probes
for MCP-1/CCL2 and RANTES/CCL5, two of the four major upregulated chemokines.
For both MCP-1 and RANTES, a strong positive staining was found in the cortex
of obstructed kidneys at days 6 and 10
(Figure 4, A and E). No signals
were detected with the use of sense sequence templates as a negative control.
Strong expression of MCP-1 and RANTES mRNA was seen in the expanded
interstitial compartment of the cortex, co-localizing to interstitial
mononuclear cell infiltrates (Figure 4, C
and G). In addition, MCP-1 expression by cortical tubular
epithelial cells frequently could be detected, whereas only a few tubular
cells were positive for RANTES mRNA (Figure
4, B and F). No MCP-1 or RANTES signals could be detected in
glomeruli. Immunohistochemical staining with antipeptide antiserum against
murine MCP-1 revealed a strong MCP-1 protein expression in interstitial cells
and positive staining in cortical tubules
(Figure 5A). Similar results
were obtained for RANTES protein expression with the use of the polyclonal
rabbit anti-mouse antibody (Figure
5B). Compared with detected mRNA transcripts by in situ
hybridization, more tubular cells showed a positive immunostaining for MCP-1
and RANTES protein. As chemokines bind to the cellular glycocalyx and to
extracellular matrix, the distribution detected by immunohistology could be
broader than that by in situ hybridization. As for MCP-1 and RANTES
mRNA transcripts, no protein could be detected in glomeruli by
immunostaining.
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Increased mRNA Expression of CCR1, CCR2, and CCR5 Chemokine Receptors
in Obstructed Kidneys
RPA revealed minimal expression of CC chemokine receptors CCR1 (binding
RANTES/CCL5 and MIP-1/CCL3), CCR2 (binding MCP-1/CCL2), and CCR5
(binding RANTES/CCL5 and MIP-1/CCL3) in contralateral kidneys 2, 6, and
10 d after UUO (Figure 6). In
obstructed kidneys, CCR1, CCR2, and CCR5 mRNA expression increased
progressively from days 2 to 10. At 2 d, no significantly increased expression
was noted compared with control kidneys. Six and 10 d after UUO, the induction
of mRNA expression was 3.6-fold and 8.4-fold for CCR1, 3.6-fold and 12.7-fold
for CCR2, and 4.4-fold and 12.8-fold for CCR5, respectively, compared with
transcript levels in contralateral kidneys. No mRNA expression of CCR1b, CCR3,
or CCR4 was detected in obstructed or contralateral kidneys at any time point
(Figure 6).
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Interstitial Localization of Murine CCR2 and CCR5 mRNA
In situ hybridization with murine CCR2 and CCR5 antisense probes
revealed an exclusive interstitial mRNA expression of the two chemokine
receptors in the renal cortex of obstructed kidneys 6 and 10 d after UUO
(Figure 7, A and B). No
specific hybridization signals could be detected in contralateral kidneys,
which showed a background signal comparable to the results after hybridization
with the sense probes (not shown). In obstructed kidneys, interstitial mRNA
expression of CCR2 and CCR5 was restricted to infiltrating mononuclear cells.
Tubular epithelial cells at sites with tubular damage, atrophy, and
interstitial mononuclear cell infiltrates or in areas with absence of severe
interstitial inflammation showed no detectable CCR2 or CCR5 expression
(Figure 7, A and B). CCR2 and
CCR5 expression was absent in endothelial cells and smooth muscle cells of the
vascular compartment. No glomerular CCR2 or CCR5 mRNA transcripts could be
detected besides occasional positive cells, which most likely reflect
circulating leukocytes in glomerular capillaries. When in situ
hybridization was combined with immunostaining for CD3 on the same section,
the majority of CD3+ lymphocytes expressed CCR5 mRNA, whereas no clear
co-localization of CD3+ cells with CCR2 mRNA transcripts could be found
(Figure 7, C and D). Thus,
infiltrating lymphocytes in the interstitial compartment seemed to express
predominantly CCR5 but not CCR2.
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CCR2 and CCR5 Expression on Infiltrating Leukocytes
We were unsuccessful in obtaining specific immunohistology for CCR2 and
CCR5 with our antibodies on tissue sections. The antibodies are, however,
suitable for FACS analysis. Therefore, to characterize further the expression
of CCR2 and CCR5 chemokine receptors on the interstitial mononuclear cell
infiltrate, we performed a four-color flow cytometry analysis. Renal cells
including inflammatory leukocytes were isolated from obstructed kidneys and
contralateral control kidneys 10 d after UUO. CCR2 and CCR5 expression was
detected on CD11b+ macrophages from obstructed kidneys
(Figure 8). Although incubation
with an irrelevant IgG2b antibody as isotype control gave a relative high
background fluorescence on the activated macrophages, a clear shift of the
mean fluorescence could be seen after staining with the specific antibodies
directed against murine CCR2 (mean fluorescence, 1267 arbitrary units
versus isotype control 520) and CCR5 (1075 versus 520). The
few macrophages isolated from contralateral control kidneys showed a similar
expression of CCR2 and CCR5 receptors
(Figure 8). In contrast,
peripheral blood monocytes of the same mice expressed high levels of CCR2
(mean fluorescence, 680 versus isotype control 222) but only moderate
amounts of CCR5 (348 versus 222)
(Figure 8). Thus, infiltrating
renal macrophages express CCR2 and CCR5 chemokine receptors after UUO.
Analysis of CD4+ and CD8+ lymphocytes isolated from obstructed kidneys
revealed a high expression of CCR5 on both lymphocyte subsets: 43.1% of CD4+
and 93.1% of CD8+ cells stained positive for CCR5
(Figure 8B), confirming the
results obtained by combined CD3 immunohistology and in situ
hybridization. In contrast, only a weak CCR2 expression could be detected on
infiltrating CD4+ or CD8+lymphocytes (10.6% of CD4+ cells and 6.1% of CD8+
cells showed a positive staining; Figure
8A). A similar pattern of CCR2 and CCR5 expression was detectable
on the few CD4+ and CD8+ lymphocytes isolated from contralateral control
kidneys. However, a lower percentage of cells expressed CCR5, and slightly
more CD4+ lymphocytes from contralateral kidneys were positive for CCR2
(Figure 8). In the peripheral
blood only 5.4% of CD4+ lymphocytes expressed CCR2, and only 5.9% expressed
CCR5. Nine and 16.2% of CD8+ lymphocytes were positive for CCR2 and CCR5,
respectively (Figure 8). These
results indicate that CCR5-positive lymphocytes of both the CD4+ and CD8+
subtype accumulate preferentially in the inflamed renal interstitium after 10
d of UUO compared with their frequency in the peripheral blood, whereas only a
small number of infiltrating lymphocytes express CCR2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In obstructed kidneys, we found a progressive increase of mRNA coding for
the CC chemokines MCP-1/CCL2, RANTES/CCL5, and for the CXC chemokines MIP-2
and IP-10/CXCL10 during the course of UUO. To a lesser extent, increased
expression of the CC chemokines eotaxin/CCL11, MIP-1/CCL3, and
MIP-1ß/CCL4 and of the C chemokine lymphotactin/XCL1 was noted. By in
situ hybridization and immunohistochemistry, we demonstrated expression
of the two major upregulated CC chemokines, MCP-1 and RANTES, in tubular
epithelial cells (predominantly MCP-1) and a very strong expression of both CC
chemokines in the interstitial compartment, co-localizing to interstitial
mononuclear cell infiltrates. Either resident interstitial cells and/or
infiltrating leukocytes may produce MCP-1 and RANTES
(31,32,33,34).
MCP-1 is a potent chemoattractant for monocytes/macrophages, and RANTES
attracts both lymphocytes and monocytes, which are a prominent histologic
feature in the UUO model. Increased expression of MCP-1 and RANTES has been
described in various forms of experimental
(8,11,12,24,35)
and human glomerulonephritis
(36,37,38),
as well as in secondary tubulointerstitial disease
(13,18,19).
MCP-1 and RANTES expression could be demonstrated in tubular epithelial cells
and, in some studies, in the interstitial compartment by in situ
hybridization or immunohistochemistry. In a rat model of puromycin
aminonucleoside—induced tubulointerstitial nephritis, MCP-1 and IP-10
mRNA production was localized to intrinsic tubulointerstitial cells
(39). In experimental
hydronephrosis, an increase in MCP-1 mRNA and protein expression was
demonstrated
(40,41),
and in a human biopsy study of congenital obstructive nephropathy,
Grandalliano et al.
(42) located MCP-1 mRNA
expression to tubules and infiltrating mononuclear interstitial cells. The
present study confirms the expression of MCP-1 and RANTES by tubular
epithelial and interstitial cells in obstructive nephropathy. Increasing
chemokine mRNA levels correlated with the extent of tubular damage,
progressive interstitial infiltration of macrophages and lymphocytes, and
concomitant fibrosis. These data suggest that locally secreted MCP-1 and
RANTES are important chemoattractant mediators of interstitial leukocyte
infiltration during the course of obstructive nephropathy.
To strengthen this hypothesis, we studied simultaneous expression of the
respective CC chemokine receptors during UUO. Indeed, we could demonstrate
that leukocytes infiltrating obstructed kidneys differentially expressed MCP-1
and RANTES receptors. Increasing levels of mRNA coding for chemokine receptors
CCR1 (binding MIP-1 and possibly murine RANTES), CCR2 (binding MCP-1),
and CCR5 (binding RANTES, MIP-1, MIP-1ß) correlate with
progressive interstitial mononuclear cell infiltrates, chemokine expression,
and fibrotic changes in obstructed kidneys. Expression of CCR2 and CCR5 mRNA
transcripts was confined strictly to the interstitial mononuclear cell
infiltrate in the cortex, as demonstrated by in situ hybridization.
Tubular epithelial cells, endothelial cells, or glomerular cells did not
express CCR2 or CCR5, confirming our previous results in mice and in human
renal biopsies
(35,43).
We and others recently found a similar upregulation of CCR1, CCR2, and CCR5 in
murine nephrotoxic serum nephritis
(35,44)
and a glomerular expression of these receptors in experimental immune-complex
glomerulonephritis (24),
whereas no CCR1b, CCR3, and CCR4 mRNA was detectable. Similarly, receptor
induction was associated with the expression of corresponding chemokine mRNA
for MCP-1 and RANTES in these and the UUO model. In both immune-complex
nephritis and obstructive nephropathy, chemokine receptor expression was
localized strictly to mononuclear cell infiltrates at the respective
glomerular (immune-complex nephritis) or tubulointerstitial (UUO) sites of
renal damage, where the corresponding chemokine production occurred.
Combination of CD3 immunostaining and in situ hybridization revealed expression of CCR5 but not CCR2 on interstitial lymphocytes. FACS analysis of cells from the UUO kidneys confirmed that infiltrating CD4+ and CD8+ lymphocytes expressed mainly CCR5, but only a few T cells were CCR2 positive. In contrast, infiltrating macrophages were positive for both CCR2 and CCR5. Peripheral blood leukocytes showed a different pattern of CCR2 and CCR5 expression: compared with kidney macrophages, circulating monocytes were positive for CCR2 but expressed only low amounts of CCR5. Circulating CD4+ and CD8+ lymphocytes expressed CCR2 and CCR5 receptors with relative low abundance, which is in striking contrast to the strong CCR5 expression on infiltrating lymphocytes. These data indicate that leukocyte subpopulations infiltrating inflamed kidneys show a distinct expression pattern of CC chemokine receptors. Interestingly, by flow cytometry, the few isolated macrophages and lymphocytes from contralateral unobstructed kidneys showed a similar expression of CCR2 and CCR5 receptors as infiltrating cells in obstructed kidneys, with a higher proportion of CD4+ lymphocytes being positive for CCR2 and a somewhat lower proportion of CD8+ lymphocytes expressing CCR5. However, this pattern clearly is different from the receptor expression on circulating blood leukocytes. These data and the uniform induction of CC chemokine receptors in different forms of nephritis suggest a distinct inflammatory phenotype of infiltrating monocytes/macrophages (expressing CCR2 and CCR5) and T lymphocytes (expressing predominantly CCR5). The differential expression of CCR2 and CCR5 on particular leukocyte subsets may allow their specific accumulation in kidney tissue. Such a tissue-specific homing has been reported for CCR4- and CCR9-positive memory T cells, which accumulate exclusively at cutaneous or intestinal sites of inflammation, respectively (44). Conversely, an accumulation of CCR2- and/or CCR5-positive mononuclear cells has been described in other organs during chronic inflammatory disease, such as arthritis, multiple sclerosis, and inflammatory bowel disease (46,47,48). Thus, the increased expression of CCR2 and CCR5 by infiltrating leukocytes also may be the result of a local modulation of chemokine receptor expression in chronically inflamed tissue compartments.
During the course of UUO, we found a strong correlation of upregulated chemokines, infiltration of CCR2- and CCR5-positive leukocytes, and progressive interstitial fibrosis, as revealed by the interstitial accumulation of FSP1+ activated fibroblasts and increased type IV collagen deposition. Infiltrating tubulointerstitial leukocytes, especially activated macrophages, are an important source of inflammatory and profibrogenic cytokines such as transforming growth factor-ß (TGF-ß). An increased tubulointerstitial expression of TGF-ß has been reported during UUO (6). Moreover, the release of CC chemokines, particularly MCP-1/CCL2, by infiltrating macrophages and lymphocytes itself may have a direct fibrogenic effect. MCP-1 has been reported to stimulate collagen and TGF-ß expression by fibroblasts, the latter resulting in an autocrine upregulation of collagen production (49). Taken together, infiltrating mononuclear cells may contribute significantly to interstitial fibrotic changes in chronic tubulointerstitial nephritis.
In summary, we demonstrated an increased expression of the CC chemokines MCP-1/CCL2 and RANTES/CCL5 at sites of progressive tubulointerstitial damage and a simultaneous interstitial accumulation of infiltrating macrophages and T lymphocytes differentially expressing the respective receptors CCR2 and CCR5 in murine obstructive nephropathy. These data suggest that CCR2- and CCR5-positive monocytes as well as CCR5-positive lymphocytes are attracted by locally released MCP-1 and RANTES, resulting in chronic interstitial inflammation. Lymphocytes that bear the CCR5 receptor may be attracted preferentially by RANTES, whereas CCR2- and CCR5-positive monocytes/macrophages could be attracted by both MCP-1 and RANTES. Blocking the ligand/receptor interaction between MCP-1 and CCR2 or RANTES and CCR5 may offer a new therapeutic approach for tubulointerstitial renal disease and progressive fibrosis.
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