By
¶
¶
From the * Department of Medicine, Boston University Medical Center, Boston, Massachusetts
02118; The Center for Blood Research, Incorporated and Department of Genetics and the § Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115;
Glaxo Institute
for Molecular Biology, Geneva, CH1228 Switzerland; and ¶ Millennium Pharmaceuticals Inc.,
Cambridge Massachusets 02139
The involvement of chemokines in inflammation is well established, but their functional role in disease progression, and particularly in the development of fibrosis, is not yet understood. To investigate the functional role that the chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES play in inflammation and the progression to fibrosis during crescentic nephritis we have developed and characterized a murine model for this syndrome. Significant increases in T-lymphocytes and macrophages were observed within glomeruli and interstitium, paralleled by an induction of mRNA expression of MCP-1 and RANTES, early after disease initiation. Blocking the function of MCP-1 or RANTES resulted in significant decreases in proteinuria as well as in numbers of infiltrating leukocytes, indicating that both MCP-1 and RANTES (regulated upon activation in normal T cells expressed and secreted) play an important role in the inflammatory phase of crescentic nephritis. In addition, neutralization of MCP-1 resulted in a dramatic decrease in both glomerular crescent formation and deposition of type I collagen. These results highlight a novel role for MCP-1 in crescent formation and development of interstitial fibrosis, and indicate that in addition to recruiting inflammatory cells this chemokine is critically involved in irreversible tissue damage.
Rapidly progressive glomerulonephritis (GN)1 is characterized by glomerular inflammation and the formation of glomerular crescents, composed of a pleiomorphic
infiltrate of mononuclear inflammatory cells and proliferating parietal epithelial cells. These crescents encroach on the
urinary space and compress the glomerular tuft causing
acute renal failure. This process is almost always associated with severe interstitial and periglomerular inflammation.
Spontaneous recovery is rare. More typically, the inflammatory infiltrate gives way to a progressive fibrotic process
involving the crescents and the periglomerular and peritubular interstitium, accompanied by tubular atrophy and
progressive renal failure. Indeed, clinical studies of patients
with glomerular diseases have shown a close correlation between the degree of interstitial fibrosis and the likelihood of
chronic renal failure (1, 2).
Although the pathogenesis of crescentic glomerulonephritis is incompletely understood and likely involves several
convergent pathways, there is general agreement that circulating mononuclear phagocytes play a central role (3, 4). Administration of nephrotoxic serum to rats results in a severe
proliferative and necrotizing GN that is characterized by
glomerular crescent formation and accumulation of leukocytes (5). These infiltrating cells may then release inflammatory mediators that influence the behavior of glomerular, tubular, and interstitial cells. This interaction between
infiltrating and resident cells leads to cellular proliferation, matrix expansion, and may ultimately lead to glomerular
sclerosis and interstitial fibrosis.
Chemokines are a superfamily of small proteins that are important in recruiting and activating leukocytes during inflammation (6). The chemokines RANTES (regulated upon
activation in normal T cells expressed and secreted) and
monocyte chemoattractant protein-1 (MCP-1) attract mainly
T lymphocytes and mononuclear phagocytes, respectively
(7, 8). In vivo, stimulated renal cells are capable of generating both of these chemokines (9, 10, 11), and both have
been found to be expressed during models of renal injury
(12, 13, 14). Several other chemokines including macrophage inflammatory protein-2 (MIP-2), platelet factor 4, interferon-inducible protein 10 kD (IP-10), and cytokine-
induced neutrophil chemoattractant (CINC), have been
demonstrated to precede the initial neutrophil influx in experimental anti-GBM GN (15, 16). Studies with neutralizing antibodies show that inhibition of CINC, MIP-2, or
MCP-1/JE activity decreases neutrophil accumulation with
an associated decline in proteinuria during the first 24 h of
disease (17). However, all of these studies concentrate on
the acute phase of the disease occurring within the first 24 h
of the treatment. Moreover, all of these studies have focused on glomerular changes and do not explore the role
that interstitial cells are likely to play in the development of
disease throughout the whole cortex. Interstitial cells are
important mediators of inflammatory and fibrotic pathways,
and the factors that they secrete are likely to be integrally
involved in the development of pathology during nephritis
(18). Therefore, the in vivo role of chemokines in both
disease induction and development remains to be established. In particular, the role of chemokines in the progression from inflammation to fibrosis is unclear.
In this study, we have developed and characterized a
murine model of accelerated nephrotoxic serum nephritis,
determined the nature of the leukocytic infiltration, and
analyzed the expression of chemokines during the progression of acute glomerular injury to glomerular crescent formation and renal fibrosis. In addition, we have determined
the functional role of the Preparation of Nephrotoxic Serum.
Nephrotoxic serum (NTS)
was prepared by immunizing male sheep with a lysate of rat glomeruli, isolated by differential sieving as described previously (19).
The sheep serum was heat inactivated at 56°C, absorbed with rat
red blood cells and serum proteins, and then filter sterilized. As
reported elsewhere (20), NTS has moderate reactivity to type IV
collagen and laminin, and substantial reactivity to glomerular cell
membrane proteins, particularly Induction of Crescentic Nephritis.
The protocol for inducing
crescentic glomerulonephritis in mice was that of Morley and
Wheeler (21), modified for our NTS to avoid rapid lethality from
acute renal failure while ensuring adequate tissue injury for analysis. CD1 mice weighing ~30 g were pre-immunized by subcutaneous injection of 200 µg normal sheep IgG in Freund's complete adjuvant. After 5 d, experimental mice were injected
intravenously with 50 µl NTS on three consecutive days and
controls were injected with 50 µl normal sheep serum (NSS) on the
same schedule. At various intervals from 12 h to 3 wk after the
first dose of NTS, groups of 6 mice were killed by ether inhalation after an overnight collection of urine in single animal metabolism cages. During this collection, mice were allowed free access
to water but not food. At the time of death, serum was obtained
by cardiac puncture, and portions of kidney were snap frozen for
immunohistology and RNA extraction, or fixed in 4% paraformaldehyde for in situ hybridization or in buffered formalin for routine histology.
Blocking Experiments.
Experiments were performed to modulate disease by the administration of mediators that neutralize or
modify chemokine function. Thus in separate experiments, groups
of mice (n = 6) received daily intravenous doses of either the
RANTES antagonist, MetRANTES (22) or hamster anti-MCP1/JE (23) 30 min before administration of 50 µl of either NTS or
NSS. MetRANTES was administered at either 16 or 32 µg per
mouse per day, and anti-MCP-1/JE was given at 5 µg per mouse
per day. Controls included mice given sterile PBS or 5 µg normal
hamster immunoglobulin (Jackson ImmunoResearch Labs., West
Grove, PA), respectively. The blocking reagents were given to
mice in the stated doses from day 0 to day 6. Mice were then
killed on day 7, after 24 h in metabolic cages, and kidneys were
removed for morphological and immunohistological analyses, as
described below.
Morphological Analysis.
Kidney halves were fixed overnight at
4°C in 10% neutral buffered formalin (Fisher), embedded in paraffin, sectioned at 3 µm and stained with hematoxylin and eosin following standard techniques. Sections from NTS mice were examined for glomerular hypercellularity and necrosis, mesangial
thickening, formation of glomerular crescents, interstitial infiltrates, and development of glomerular and interstitial fibrosis, as
compared to NSS mice. A crescent score was obtained by counting the number of glomeruli showing cellular crescents in at least
100 glomeruli per mouse.
Assessment of 24 h Proteinuria.
Urine protein excretion was
measured on timed overnight specimens collected at intervals from
3 d to 3 wk from individual mice in metabolism cages and assayed
by the sulphosalicylic method (19). Blood urea nitrogen was measured on 200-µl serum samples by the Boston University Medical
Center clinical laboratories using an auto-analyzer.
Immunohistochemical Phenotyping and Quantitation of Leukocytes.
Kidneys from treated and untreated mice were excised,
rolled in Tissue Tek OCT compound (Cryoform, IEC, Needham,
MA) snap frozen in liquid nitrogen and stored at Measurement of mRNA Expression by Northern Blots.
Total RNA
from kidneys obtained from NTS- and NSS-treated mice was
isolated using the guanidinium thiocyanate/acid phenol procedure (24). RNA was extracted from PBS-treated mice at the same time points as NTS and NSS mice to determine basal levels of chemokines. Northern blots (25) were prepared with 20 µg of
total RNA, purified as indicated above, fractionated in a 1.5% agarose/formaldehyde gel and blotted onto a nylon membrane
(Genescreen, DuPont). Membranes were probed using 32P-labeled
probes for RANTES (26), MCP-1/JE (27), MIP-1 Collagen I Deposition.
Samples of frozen kidney from NTS-
and NSS-treated mice were sectioned at 4 µm, washed in PBS,
fixed in acetone, and incubated with polyclonal rabbit anti-
mouse type I collagen (Chemicon International, Temecula, CA).
The primary antibody was detected with goat anti-rabbit IgGFITC (Jackson ImmunoResearch Labs.). Both the primary and
secondary antibodies were diluted to their optimal working concentration in 2% sheep IgG in PBS. The effectiveness of this
blocking procedure was confirmed by a negative staining reaction
when a non-specific normal rabbit serum was substituted for the
primary antibody. Tissues were also stained for sheep and mouse
IgG and mouse C3 using various commercially available FITCconjugated antisera. Sections were imaged using NIH image 1.56, with 10 randomly chosen fields per section (at a magnification of
×20) being screened to calculate the mean area stained for each
section.
Collagen I RNA Expression.
These studies used a 600-bp cDNA
probe that codes for the COOH terminus and a small part of the
untranslated region of the rat Statistical Analysis.
The significance of differences between
experimental groups was calculated by the Wilcoxon MannWhitney test using Xlstat in Microsoft Excel 5.
The severity of glomerulonephritis was dose dependent and varied from mild
glomerular hypercellularity to anuric acute renal failure and
early death from extensive glomerular necrosis and crescent
formation. There were no differences in histological appearance of kidneys from NSS mice compared with mice
given PBS only (Fig. 1 A). 12 h after the first injection of
NTS, proteinaceous casts were present in renal tubules, coincident with the onset of proteinuria. After 24 h, leukocytes were evident within the interstitium and by day 3 prominent perivascular infiltrates were present (Fig. 1 B).
At this point the glomeruli were clearly hypercellular with
focal areas of necrosis. On day 5 some glomeruli contained crescents and interstitial and perivascular infiltrates were
pronounced. On day 7, 55% of glomeruli exhibited prominent cellular crescents (Fig. 1 C) and marked abnormalities
were present within the tubules and interstitium. These included severely dilated tubules with flattened or denuded
epithelia and expansion of the interstitium due to edema,
interstitial infiltrates, and the onset of fibrosis (Fig. 1 D). On
day 14, the fibrotic process was more diffuse throughout the interstitium and had extended to involve some of the
glomerular crescents, apparently by invasion through Bowman's capsule.
Mice injected with NTS were
significantly proteinuric by day 3 and became progressively
more so throughout the course of the study (Fig. 2 A).
Blood urea nitrogen levels rose rapidly during the acute
phase of immunologic injury but then improved towards baseline values over the course of 10 d (Fig. 2 B). However, concomitant with the development of progressive interstitial and glomerular fibrosis, blood urea nitrogen levels
rose progressively from day 14.
Over the course of the disease,
both the interstitium and crescents became increasingly fibrotic as demonstrated by Masson trichrome staining (not
shown) and deposition of type I collagen (Fig. 3). Most
strikingly, a rapid induction of cells expressing mRNA for
Infiltrating lymphocytes, neutrophils and mononuclear phagocytes were
identified at different time points during the development of crescentic GN by immunohistochemical staining of frozen sections. (Fig. 4). Increases in both neutrophils and
mononuclear phagocytes, but not lymphocytes, were observed 12 h after the first injection of NTS. However, by
day 3, all three cell types were increased in kidneys from
NTS mice when compared with those from NSS mice at
the same time period. Maximal numbers of neutrophils
were observed at day 3 of disease, whereas lymphocyte
numbers continued to be raised even at day 14. Peak macrophage infiltration was seen at day 7 with a decline in numbers occurring by day 14.
Expression of chemokines involved
in the recruitment of leukocytes to sites of inflammation
was determined by Northern analysis of RNA isolated
throughout the development of crescentic GN (Fig. 5).
Therefore, mRNA expression for RANTES (CD4+ T cell
and eosinophil chemoattractant) (32); MCP-1/JE (monocyte chemoattractant) (33); MIP-1
MetRANTES was
used to neutralize the effect of this chemokine in order to
define the functional role that RANTES plays in the development of crescentic GN. Day 7 was chosen to determine the effect of blocking RANTES as by this time there is a
significant infiltration by leukocytes, an increase in proteinuria, and the beginning of the development of fibrosis, as
marked by collagen I deposition. Thus, 16 µg MetRANTES
was administered daily until day 6, when mice were housed
in metabolic cages for 24 h before killing on day 7. Four
disease parameters were then assessed for the effect of MetRANTES: 24 h proteinuria, leukocyte infiltration, glomerular
crescent formation and collagen deposition. First, MetRANTES was seen to induce a 36% decrease in excretion of
protein (31 ± 5 mg/24 h in treated versus 52 ± 7 mg/24 h in
untreated NTS mice, P <0.05) (Fig. 6 A). In conjunction
with this there was a 52% decrease in numbers of T cells and
a 40% decline in mononuclear phagocyte numbers within
sections (P <0.005). However, there was no difference in
numbers of neutrophils after MetRANTES treatment (Fig. 6
B). A 16% decrease in glomerular crescent formation was
counted in H&E stained sections (not significant), although no other changes in morphological abnormalities was seen
to occur (Fig. 6 C). There was no difference in the deposition
of type I collagen after administration of MetRANTES (Fig. 6
D). This experiment was repeated with a higher dose of MetRANTES (32 µg/mouse/d) without additional benefit (data
not shown).
A neutralizing monoclonal antibody specific for MCP-1/JE was used
to assess the contribution that this chemokine makes to the
development of crescentic GN. Experiments were conducted exactly as described above for MetRANTES, with
5 µg of anti-MCP-1/JE or 5 µg hamster immunoglobulin
G (hIgG) being administered daily from day 0 to day 6 to
NTS or NSS mice. Hamster Ig had no effect upon the development of disease. Proteinuria was seen to diminish by
25% upon administration of anti-MCP-1/JE (P <0.01)
(Fig. 7 A). Immunohistochemical phenotyping revealed that
neutralizing MCP-1/JE resulted in a 47% decrease in macrophages within both glomerular and the interstitial areas
(P <0.05). Similarly, there was a decrease in numbers of
lymphocytes (P <0.05). Interestingly, neutralizing MCP1/JE enhanced the increase in numbers of neutrophils, by
37% compared to untreated NTS mice (P <0.01)(Fig. 7 B).
Interestingly there was a striking decrease (by 73.5%) in the
number of glomerular crescents on day 7 in treated mice compared to untreated NTS mice (P <0.001) (Fig. 7 C). Concomitant with this there was a 65% decrease in the deposition
of type I collagen in sections from anti-MCP-1-treated NTS mice compared to untreated NTS mice (P <0.005)
(Fig. 7 D).
In this study we have investigated the role of chemokines in the development of glomerular and interstitial inflammation and progression to renal fibrosis in a murine
model of crescentic glomerulonephritis. Glomerular and interstitial hypercellularity are the earliest abnormalities in this
model of accelerated nephrotoxic serum nephritis (Fig. 1),
and they occur concomitantly with the appearance of severe proteinuria and transient acute renal failure (Fig. 2). The
inflammatory infiltrate in glomeruli and interstitium is
composed of T-lymphocytes, mononuclear phagocytes and
neutrophils (Fig. 4), much as has been described in other
animal models of crescentic glomerulonephritis (34). Within
one week from the onset of disease, glomerular crescents
begin to form and there is perivascular and periglomerular
type I collagen gene induction (Fig. 3), signifying the onset
of renal fibrosis. PMNs, mononuclear phagocytes and lymphocytes may contribute to acute renal cell injury and progression of glomerular disease by a variety of different mechanisms including inducing the secretion of pro-inflammatory mediators such as TNF To determine the role of chemokines in eliciting the migration of inflammatory cells to the kidney in this model,
we first examined their expression during the evolution of
the lesion and then established the functional role of likely
candidates with blocking agents. RANTES and MCP-1 are
strongly expressed after disease induction (Fig. 5) at a time
that coincides with the influx of T-lymphocytes and mononuclear phagocytes, and that follows the earlier appearance
of neutrophils. MIP1 Progression of disease in mice with crescentic GN was
marked by the development of glomerular crescents and the
deposition of type I collagen within the interstitium. We
have examined the role that MCP-1 and RANTES play in
the progression to this later stage of disease. Although administration of a RANTES antagonist resulted in a decrease in the accumulation of T cells and monocytes/macrophages, there was little effect on glomerular crescent
formation or deposition of interstitial type I collagen. Previous studies have suggested that activated periglomerular
T cells may be involved in the disruption of Bowman's
capsule (36), and CD8+ T cell depletion has been found to
inhibit crescent formation, (37), but we have shown that
RANTES does not appear to be involved in this process.
Conversely, neutralizing antibodies to MCP-1 resulted in a
striking decrease in both glomerular crescent formation and
collagen I deposition. It is interesting that although macrophage accumulation was only inhibited by 50% by antibody administration, this decrease is sufficient to cause such
an impressive effect on markers of fibrosis. Since monocyte/macrophage accumulation was not completely inhibited by MCP-1 neutralization, it seems likely that other
macrophage-specific chemokines may contribute to this
migration. It is not inconceivable that MCP-1 not only affects migration and extravasation of mononuclear phagocytes but mediates direct effects upon the resident renal
cells, which in turn affects the pathway to fibrosis. MCP-1
may participate in this fibrotic process directly, by inducing
the secretion of extracellular matrix components, or indirectly by instigating the secretion of other pro-fibrotic mediators. It is also possible that MCP-1 alters the phenotype
of resident renal cells leading to parietal glomerular epithelial cell proliferation, collagen I production by interstitial fibroblasts, or even tubular epithelial cell transmodulation
(38, 39). Whether this is a direct effect of MCP-1, or is
mediated by other fibrogenic agents such as TGF- This work has centered on characterizing a murine
model of crescentic nephritis and determining the role of
the chemokines MCP-1 and
RANTES in the development of glomerular and interstitial
inflammation and crescent formation, as well as in the evolution of the ensuing fibrosis.
1 integrin and its accompanying
chain.
70°C. 4-µm
cryosections were cut onto microscope slides, air dried, and then
fixed in acetone at 4°C for 20 min. Fixed sections were stained
with rat monoclonal antibodies directed against Thy1 (T cells),
GR-1 (granulocytes) (both PharMingen, San Diego, CA) and
MOMA-2 (mononuclear phagocytes) (Biosource International, Camarillo, CA) using an avidin/biotin staining method. All incubations were carried out under humidified conditions and slides were
washed twice between steps for 5 min each in 0.1 M PBS. First,
endogenous peroxidase was blocked by incubation for 20 min in
methanol containing 0.3% hydrogen peroxide. Nonspecific staining due to crossreaction with endogenous avidin or biotin was
then blocked by incubation with avidin solution followed by biotin solution, both for 20 min (Vector, Burlingame, CA). Thereafter, sections were overlaid with 20% normal rabbit serum in
PBS for 15 min and then incubated overnight at 4°C with mAbs
specific for either Thy1, MOMA-2, or GR-1 (all at 1/10 in 10%
normal mouse serum in PBS) (PharMingen). Bound monoclonal antibody was visualized by incubation with biotinylated rabbit anti-rat immunoglobulin diluted 1/200 in 10% normal mouse serum in PBS, and then streptavidin peroxidase complex (prepared
according to manufacturer's instructions) for 30 min each (both
Dako, Carpinteria, CA). Finally, slides were flooded with peroxidase substrate solution (400 mg diaminobenzidine in 10 ml of
PBS, containing 0.01% hydrogen peroxide) for 10 min. Control
slides were included where monoclonal antibody, biotinylated
anti-rat immunoglobulin or streptavidin complex were selectively
omitted. All slides were counterstained with hematoxylin. For
each antibody the number of positively stained cells was determined in 15 high-power fields chosen at random per section
(40× magnification; total area 1.8 mm2) and the average number
of cells/field was calculated for each mouse.
(28), and
TCA-3 (29) applied in 50% formamide hybridization solution at
42°C for 18 h. Blots were washed in 2× SSC/1%SDS at 45°C
and exposed at
70°C on Kodak XAR5 film (Sigma).
1 type I collagen gene (30) as previously described (31). The cDNA was inserted into and propagated in a pBluescript vector (Stratagene) carrying flanking T3
and T7 RNA polymerase promoters. Once linearized, either of the
flanking RNA polymerase promoters could be employed to create radioactively labeled sense or anti-sense riboprobes by a runoff
transcription method (25). Runoff transcription products were
hydrolyzed in 0.01 M DTT, 0.08 M NaHCO3, 0.12 M NaCO3
for 60 min at 60°C and neutralized in 0.01 M DTT, 0.2 M Na
acetate, 0.17 M acetic acid. The product was recovered by salt- ethanol precipitation and Sephadex G-50 chromatography (Pharmacia, Piscataway, NJ). The fractions of interest were pooled, the
volume adjusted to give a final specific activity of 3 × 106 cpm/
ml in 0.3 M DTT, and stored at
70°C until used. Paraffin sections of 4-µm thickness were cut onto Superfrost®/Plus slides
(Fisher Scientific, Pittsburgh, PA), dewaxed, and hydrated. Prehybridization, hybridization, and post-hybridization procedures
were carried out exactly as previously described (31). Finally, the
sections were coated with Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) and sealed in light-tight boxes at 4°C for 7-14 d. The slides were developed with Kodak D-19 (Eastman Kodak Co.), counterstained with Mayer's hematoxylin and
mounted using a water soluble mountant. Specificity was evaluated by use of the sense riboprobe or by introduction of an
RNAse incubation step during the prehybridization. Sections
were examined and photographed with a Nikon Optiphot microscope equipped for dark-field illumination at 100× magnification and bright-field illumination at 100× and 400× magnification (Nikon, Garden City, NY).
Morphological Changes in Renal Tissue.
Fig. 1.
Administration of
nephrotoxic serum to pre-immunized mice results in a severe
proliferative and necrotizing GN.
Hematoxylin and eosin staining
of kidney sections from mice
given either NSS (A) or NTS
(B-D). NTS mice showed glomerular and interstitial infiltrates
(B); glomerular crescent formation (C and D). Arrows highlight
crescentic glomeruli. Original
magnification: (A, B, and D)
×40; (C) ×20.
[View Larger Version of this Image (200K GIF file)]
Fig. 2.
Nephrotoxic serum results in a decline in renal function. Renal dysfunction in mice as shown by (A) proteinuria and (B) blood urea
nitrogen. Each point represents the mean values from either NSS (open circles, n = 6) or NTS (closed circles, n = 15) treated mice, and bars show the
standard error of the mean.
[View Larger Version of this Image (16K GIF file)]
1 type I collagen was seen by in situ hybridization (ISH)
in perivascular, periglomerular and intervening regions of
cortex within 3 d of the final injection of NTS (Fig. 3, A and C). These cells differed from those that constitutively
express
1(I) collagen in the perivascular region of normal
and control mice and they progressively increased in number, intensity of signal, and extent over the course of disease. Immunofluorescent staining showed deposition of
collagen protein in corresponding areas (Fig. 3, B and D).
Initially, glomeruli were negative for type I collagen by IF
and ISH, but with time (around day 14),
1(I) mRNApositive cells encircling the glomeruli appeared to infiltrate
the Bowman's capsule of some glomeruli and were followed by positive IF staining of the crescents for type I collagen (data not shown).
Fig. 3.
Nephrotoxic serum
induces expression of type I collagen mRNA and protein. Deposition of type I collagen in renal
tissue isolated from NSS (A and
B) or NTS (C and D) mice on
day 7 of crescentic GN. Plates
show (A and C) a 4-µm paraffin
section hybridized with a 35ScDNA probe encoding a region
of the at 1 type I collagen gene and (B and D) a 4-µm frozen
section stained for mouse type I
collagen using indirect immunofluorescence. G, glomerulus; *,
lumen of tubule; V, venule. Original magnification ×40.
[View Larger Version of this Image (111K GIF file)]
Fig. 4.
Nephrotoxic serum induces an increase in numbers of leukocytes within renal tissue. Sections were prepared from NSS (open circles) or
NTS (closed circles) mice at various times after disease induction and stained
with antibodies specific for T-lymphocytes (a), mononuclear phagocytes
(b), and neutrophils (c). Positively stained cells were counted in 15 high
power fields per section (total area = 0.5mm2). Each dot represents one
individual mouse analyzed (between 3 and 10 mice per group) with bars
depicting means for each group.
[View Larger Version of this Image (19K GIF file)]
(monocyte and eosinophil chemoattractant) (28); and TCA3 (neutrophil and
monocyte chemoattractant) (23) was assessed. There was
no expression of either MCP-1/JE or RANTES at 12 or
24 h after the induction of disease, but from day 3 onwards NTS treated mice showed strong expression of these chemokines, whereas NSS- or PBS-treated mice did not.
There was no detectable expression of TCA-3 in either
NTS- or NSS-treated mice (data not shown). Low expression of MIP-1
was detected in all mice, with no discernible difference between mice from NTS or NSS groups.
Fig. 5.
Kidneys from mice with crescentic nepritis show expression
of inflammatory chemokines. Northern blot analysis was performed using
total RNA (20 µg) extracted from kidneys isolated from NSS or NTS
mice throughout disease. Expression of two representative mice at each
time point is included. Expression of -actin was used to determine the
quality and quantity of RNA.
[View Larger Version of this Image (57K GIF file)]
Fig. 6.
MetRANTES reduces proteinuria and mononuclear cell infiltration. The effects of the RANTES antagonist MetRANTES on development of (A) proteinuria, (B) leukocyte infiltration, (C) glomerular crescent formation, and (D) deposition of type I collagen is shown. Groups of NSS (open circles, n = 3) and NTS (closed circles, n = 6) mice
given 16 µg/day of MetRANTES or PBS were scarified on day 7 of disease. For A, C, and D, each point represents one mouse and bars indicate
the mean for each group. For B, numbers of T-lymphocytes (T), mononuclear phagocytes (M) and granulocytes (G) were enumerated in immunohistochemically stained sections and bars represent the standard error of
the mean for each group. Positively stained cells were counted within the
glomeruli (open bars) and interstitium (shaded bars) of 15 randomly chosen,
high power fields within each section. For C, one hundred glomeruli were
counted in a hematoxylin and eosin-stained section from each mouse, and
the percentage of glomeruli containing cellular crescents was calculated.
In D, kidney sections were stained with an antibody against type I collagen, which was detected with a fluoresceinated secondary antibody.
Distribution of immunofluorescence was measured in 10 random fields
from each section using NIH image 1.56. For each panel the significance
of differences between treated and untreated groups of NTS mice were
determined by a Wilcoxon Mann-Whitney test.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
Antibodies to MCP-1 reduce proteinuria and mononuclear
cell infiltration, as well as glomerular crescent formation and collagen deposition. The effects of neutralizing antibodies to MCP-1/JE on (A)
proteinuria, (B) leukocyte infiltration, (C) glomerular crescent formation,
and (D) deposition of type I collagen are shown. Groups of NSS (open circles, n = 4) and NTS (closed circles, n = 10) were treated daily with 5 µg/d
hamster anti-mouse MCP-1/JE or hamster immunoglobulin and were
then killed on day 7 of disease. For A, C, and D, each point represents
one mouse and bars indicate the mean for each group. For B, numbers of
T-lymphocytes (T), mononuclear phagocytes (M), and granulocytes (G)
were enumerated in immunohistochemically stained sections and bars
represent the standard error of the mean for each group. Positively stained cells were counted within the glomeruli (open bars) and interstitium (shaded bars) of 15 randomly chosen, high power fields within each section. For C, 100 glomeruli were counted in a hematoxylin and eosinstained section from each mouse, and the percentage of glomeruli containing cellular crescents was calculated. In D, kidney sections were
stained with an antibody against type I collagen that was detected with a
fluoresceinated secondary antibody. Distribution of immunofluorescence
was measured in 10 random fields from each section using NIH image
1.56. For each panel the significance of differences between treated and
untreated groups of NTS mice were determined by a Wilcoxon MannWhitney test.
[View Larger Version of this Image (32K GIF file)]
and IL-1, which may then induce
chemokine expression by resident renal cells. The interaction between infiltrating inflammatory cells and intrinsic
renal cells may lead to cell proliferation and matrix expansion which would ultimately result in glomerulosclerosis
and interstitial fibrosis.
was found to be constitutively expressed in the kidney and was not upregulated during crescentic nephritis. We did not see any expression of TCA3
during crescentic nephritis, but this may reflect the restricted secretion profile of this particular chemokine (23).
MCP-1 and RANTES are both critical mediators in the
evolution of inflammatory reactions and are known to be
secreted by isolated resident renal cells in vitro (11, 17, 26)
as well as by infiltrating inflammatory cells. Expression
of both RANTES and MCP-1 has been documented in
several human nephritides (35) as well as during experimental anti-GBM GN (14, 17) but these studies are limited
to examining glomerular changes occurring during a relatively short time period and do not provide evidence that
chemokines are involved in the development of inflammation and disease progression. In our study however, administration of a RANTES antagonist resulted in a decrease in
the accumulation of T cells and monocytes/macrophages
concomitant with a partial attenuation of proteinuria (Fig. 6).
MetRANTES is a functional antagonist of RANTES and
blocks calcium mobilization as well as chemotaxis of T cells
and monocytes (22). Interestingly, MetRANTES antagonizes RANTES and MIP-1
with equal potency in chemotaxis assays but not calcium mobilization. It is thought to
act as a competitive inhibitor on the shared MIP-1
/
RANTES receptor. We did not find increased expression of
MIP-1
in our model of crescentic GN and therefore assume that Met-RANTES is acting primarily to antagonize
RANTES, thereby partially blocking the in vivo migration
of T cells and mononuclear phagocytes. We have also shown
that inhibition of MCP-1 by administration of neutralizing
antibodies results in decreased accumulation of mononuclear phagocytes and T-lymphocytes, concomitant with
a partial attenuation of proteinuria (Fig. 7). Thus, we have
shown that both MCP-1 and RANTES are critically involved
in the primary inflammatory phase of crescentic GN.
, PDGF, or FGF, has yet to be determined. We have preliminary data demonstrating the expression of TGF-
mRNA
in murine crescentic glomerulonephritis (data not shown),
however despite its well-established fibrogenic properties
(40), the functional role of this cytokine in post-inflammatory interstitial fibrosis of the kidney has yet to be documented. It is noteworthy that the mice in our model are
heavily proteinuric, which some have suggested may be a
sufficient stimulus for renal tubular chemokine production
and consequent interstitial inflammation and fibrosis (41).
On the other hand, proteinuria in the absence of accompanying glomerular inflammation does not appear to stimulate MCP-1 expression (41).
chemokines MCP-1 and RANTES in the renal inflammation, crescent formation and in the development of
the ensuing interstitial fibrosis. We can conclude that both
chemokines play a role in the influx of inflammatory cells
to the glomeruli and interstitium and in the initial development of renal dysfunction. Moreover, MCP-1 seems to
play a critical role in the development of the characteristic glomerular crescents and in the deposition of type I collagen. It is likely that this chemokine mediates direct effects
upon intrinsic renal cells which ultimately leads to the formation of crescents and secretion of matrix components.
Address correspondence to J.C. Gutierrez-Ramos, Millennium Pharmaceuticals Inc., 640 Memorial Drive, Cambridge, MA 02139. Any queries regarding the murine model of crescentic nephritis should be directed to Dr. D. Salant, Renal Section, Department of Medicine, 88 East Newton St., Boston, MA 02118.
Received for publication 18 December 1996.
The authors are indebted to Drs. Ramzi Cotran and Jose-Angel Gonzalo for helpful discussion while preparing this manuscript.1. | Risdon, R.A., J.C. Sloper, and H.E. DeWardener. 1968. Relationship between renal function and histological changes found in renal biopsy specimens from patients with persistent glomerulonephritis. Lancet. 2: 363-366 [Medline]. [Medline] |
2. | Bohle, A., M. Wehrmann, O. Bogenschultz, W. Vogel, H. Schmitt, C.A. Muller, and G.A. Muller. 1992. The long term prognosis of the primary glomerulonephritides. A morphologic and clinical analysis of 1747 cases. Pathol. Res. Pract. 188: 908-924 [Medline]. [Medline] |
3. | Thompson, N.M., S.R. Holdsworth, E.F. Glasgow, and R.C. Atkins. 1979. The macrophage in the development of experimental crescentic glomerulonephritis. Am. J. Pathol. 94: 223-240 . [Medline] |
4. | Schreiner, G.F.. 1991. The macrophage in glomerular injury. Seminar Nephrol 11: 268-275 [Medline]. |
5. | Sado, Y., I. Naito, M. Akita, and T. Okigaki. 1986. Strain specific responses of inbred rats on the severity of experimental autoimune glomerulonephritis. J. Clin. Lab. Immunol. 19: 193-199 [Medline]. [Medline] |
6. | Schall, T.J., and K.B. Bacon. 1994. Chemokines, leukocyte trafficking, and inflammation. Curr. Opin. Immunol. 6: 865-873 [Medline]. [Medline] |
7. | Schall, T.J., N.J. Simpson, and J.Y. Mak. 1992. Molecular cloning and expression of the murine RANTES cytokine: structural and functional conservation between mouse and man. Eur. J. Immunol. 22: 1477-1481 [Medline]. [Medline] |
8. | Rollins, B.J., T. Yoshimura, E.J. Leonard, and J. Pober. 1990. Cytokine-activated human endothelial cells synthesize and secrete monocyte chemoattractant protein, MCP-1. Am. J. Pathol. 136: 1229-1233 [Medline]. [Abstract] |
9. | Hora, K., J. Satriano, A. Santiago, T. Mori, E. Stanley, Z. Shan, and D. Schlondorff. 1992. Receptors for IgG complexes activate synthesis of monocyte chemoattractant peptide 1 and colony-stimulating factor 1. Proc. Natl. Acad. Sci. USA. 89: 1745-1749 [Medline]. [Abstract] |
10. |
Satriano, J.,
M. Schuldiner,
K. Hora,
Y. Xing,
Z. Schan, and
D. Schlondorff.
1993.
Oxygen radicals as second messengers
for expression of the monocyte chemoattractant protein JE/
MCP-1, and the monocyte colony stimulating factor, CSF-1,
in response to tumour necrosis factor-![]() |
11. |
Wolf, G.,
S. Alberle,
F. Thaiss,
P. Nelson,
A. Krensky,
E. Neilson, and
R. Stahl.
1993.
TNF-![]() |
12. | Stahl, R., F. Thaiss, M. Disser, U. Helmchen, K. Hora, and D. Schlondorff. 1993. Increased expression of monocyte chemoattractant protein-1 in anti-thymocyte antibody-induced glomerulonephritis. Kidney Int. 44: 1036-1047 [Medline]. [Medline] |
13. | Thaiss, F., U. Helmchen, G. Zahner, U. Haberstroh, N. Radounikli, W. Schoeppe, and R. Stahl. 1993. muRANTES mRNA expression in glomeruli isolated from rats with autologous nephrotoxic serum nephritis. J. Am. Soc. Nephrol. 4: 638(Abstr.). |
14. |
Tang, W.W.,
S. Yin,
A.J. Wittwer, and
M. Qi.
1995.
Chemokine gene expression in anti-glomerular basement membrane antibody glomerulonephritis.
Am. J. Physiol.
269:
F323-F330
[Medline].
|
15. | Feng, L., Y. Xia, T. Yoshimura, and C.B. Wilson. 1995. Modulation of neutrophil influx in the rat with anti-macrophage inflammatory protein-2 (MIP-2) antibody. J. Clin. Invest. 95: 1000-1017 . |
16. | Wu, X., A.J. Wittwer, L.S. Carr, B.A. Crippes, J.E. Delarco, and J.B. Lefkowith. 1994. Cytokine-induced neutrophil chemoattractant mediates neutrophil influx in immune complex glomerulonephritis in the rat. J. Clin. Invest. 94: 337-344 [Medline]. [Medline] |
17. | Tang, W.W., M. Qi, and J.S. Warren. 1996. Monocyte chemoattractant protein 1 mediates glomerular macrophage infiltration in anti-GBM Ab GN. Kidney Int. 50: 665-671 [Medline]. [Medline] |
18. | Strutz, F., and E.G. Neilson. 1994. The role of lymphocytes in the progression of interstitial disease. Kidney Int. 45: S106-S110 . |
19. | Salant, D.J., and A.V. Cybulsky. 1988. Experimental glomerulonephritis. Methods Enzymol. 162: 421-461 [Medline]. [Medline] |
20. |
O'Meara, Y.M.,
Y. Natori,
A.W. Minto,
D.J. Goldstein,
E.J. Manning, and
D.J. Salant.
1992.
Nephrotoxic antiserum identifies a ![]() |
21. | Morley, A.R., and J. Wheeler. 1985. Cell proliferation within the Bowman's capsule in mice. J. Pathol. 145: 315-327 [Medline]. [Medline] |
22. |
Proudfoot, A.,
C. Power,
A. Hoogewerf,
M.-O. Montjovent,
F. Borlat,
R. Offord, and
T. Wells.
1996.
Extension of
recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist.
J Biol Chem.
271:
2599-2603
[Medline].
|
23. |
Luo, Y.,
J. Laning,
S. Devi,
J. Mak,
T.J. Schall, and
M.E. Dorf.
1994.
Biologic activities of the murine ![]() |
24. | Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159 [Medline]. [Medline] |
25. | Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 7.1-7.87. |
26. | Heeger, P., G. Wolf, C. Meyers, M.J. Sun, S.C. O'Farrell, A.M. Krensky, and E.G. Neilson. 1992. Isolation and characterization of cDNA from renal tubular epithelium encoding murine RANTES. Kidney Int. 41: 220-226 [Medline]. [Medline] |
27. | Rollins, B.J., E.D. Morrison, and C.D. Stiles. 1988. Cloning and expression of JE, a gene inducible by platelet-derived growth factor and whose product has cytokine-like properties. Proc. Natl. Acad. Sci. USA. 85: 3738-3742 [Medline]. [Abstract] |
28. |
Widmer, U., and
v.D. Yang, S., K.R. Manogue, B. Sherry, and
A. Cerami.
1991.
Genomic structure of murine macrophage
inflammatory protein-1![]() |
29. |
Burd, P.R.,
G.J. Freeman,
S.D. Wilson,
M. Berman,
R. DeKruyff,
P.R. Billings, and
M.E. Dorf.
1987.
Cloning and
characterization of a novel T cell activation gene.
J. Immunol.
139:
3126
[Medline].
|
30. |
Genovese, C.,
D. Rowe, and
B. Kream.
1984.
Construction
of DNA sequences complementary to rat ![]() ![]() |
31. | Minto, A.W., M.A. Fogel, Y. Natori, Y.M. O'Meara, D.R. Abrahamson, B. Smith, and D.J. Salant. 1993. Expression of type I collagen mRNA in glomeruli of rats with passive Heymann nephritis. Kidney Int 43: 121-127 [Medline]. [Medline] |
32. | Schall, T.J., K. Bacon, K.I. Toy, and D.V. Goeddel. 1990. Selective attraction of monocytes and T lymphocytes of the memory phenotype by the cytokine RANTES. Nature (Lond.). 347: 669-671 [Medline]. [Medline] |
33. |
Ernst, C.A.,
Y.J. Zhang,
P.R. Hancock,
B.J. Rutledge,
C.L. Corless, and
B.J. Rollins.
1994.
Biochemical and biologic characterization of murine monocyte chemoattractant protein-1.
J. Immunol.
152:
3541-3549
[Medline].
|
34. | Atkins, R.C., D.J. Nikolic-Paterson, Q. Song, and H.Y. Lan. 1996. Modulators of crescentic glomerulonephritis. J. Am. Soc. Nephrol. 7: 2271-2278 [Medline]. [Abstract] |
35. | Lan, H.Y., D.J. Nikolic-Paterson, and R.C. Atkins. 1992. Involvement of activated periglomerular leukocytes in the rupture of Bowman's capsule and glomerular crescent progression in experimental glomerulonephritis. Kidney Int 67: 743-751 . |
36. | Kawasaki, K., E. Yaoita, T. Yamamoto, and I. Kihara. 1992. Depletion of CD8 positive cells in nephrotoxic serum nephritis of WKY rats. Kidney Int. 41: 1517-1526 [Medline]. [Medline] |
37. | El Nahas, A.M., E.C. Muchaneta-Kubara, G. Zhang, A. Adam, and D. Goumenos. 1996. Phenotypic modulation of renal cells during experimental and clinical renal scarring. Kidney Int. 49: S23-S27 . |
38. | Okada, H., F. Strutz, T.M. Danoff, and E. G. Neilson. 1996. Possible pathogenesis of renal fibrosis. Kidney Int 49: S37-S38 . |
39. |
Border, W.A., and
N.A. Noble.
1994.
Transforming growth
factor ![]() |
40. | Eddy, A.A., and J.S. Warren. 1996. Expression and function of monocyte chemoattractant protein-1 in experimental nephrotic syndrome. Clin. Immunol. Immunopathol. 8: 140-151 . |