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*
Department of Pathology, Harvard Medical School, Boston, MA 02115; and
Millennium Pharmaceuticals, Inc., Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMethods and MaterialsResultsDiscussionReferences |
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, MIP-1ß, and IFN-
-inducible protein-10 were induced
following treatment with higher doses of MIP-2 or KC. These effects are
specific to mesangial cells, as MIP-2 or KC treatment of renal cortical
epithelial cells or peritoneal macrophages failed to induce chemokine
production. This cascade of chemokine interactions may contribute to
renal infiltration and leukocyte activation. The abilities of MIP-2 or
KC to stimulate their own synthesis may also contribute to the
maintenance and chronic course of glomerular inflammation. The
mesangial cell receptor for MIP-2 and/or KC is unknown but is not
CXC-chemokine receptor-2. |
Introduction |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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The potential involvement of chemokines in the pathogenesis of various infectious and inflammatory diseases has drawn considerable attention (4, 5, 6). Immunohistochemical analysis of human kidney biopsies indicated that expression of the CC-chemokine monocyte chemoattractant protein-1 (MCP-1)3 correlated with the local infiltration of macrophages in membranous nephropathy and glomerulosclerosis (6, 7). Other studies identified glomerular staining for MCP-1 in biopsies of patients with proliferative glomerulonephritis and lupus nephritis (8, 9). These data suggest a role for MCP-1 in the macrophage infiltrate of various forms of nephritis.
Chemokines are also implicated in the pathogenesis of experimental
kidney diseases. In rodent models of Ab-induced glomerulonephritis.
mRNA for macrophage inflammatory protein-2 (MIP-2), a CXC-chemokine
that is chemotactic for neutrophils, was increased within the first
hour after induction of disease, whereas mRNA for MCP-1 appeared later
(10, 11, 12). In these models, the kinetic relationships among
infiltrating neutrophils and monocytes correlate with the appearance of
CXC- and CC-chemokines (12, 13, 14). Independent experiments
using anti-chemokine Abs have also indicated a role for CXC- and
CC-chemokines in the evolution of renal pathogenesis. Administration of
Abs directed to the CXC-chemokines MIP-2, cytokine-inducible neutrophil
chemoattractant, or IL-8 reduced acute inflammation, fibrin deposition,
and glomerular damage as evidenced by reduction of proteinuria in
rodent disease models (12, 14, 15). Similarly, treatment
with Abs against the CC-chemokine MCP-1 reduced proteinuria,
inflammation, interstitial fibrosis, and glomerular crescent formation,
but the animals developed exacerbated neutrophil infiltrates
(14). In comparison, treatment with a RANTES antagonist
inhibited proteinuria and the numbers of infiltrating leukocytes but
not fibrosis or crescent formation (14). A separate report
using an Ab-mediated nephritis model in rats demonstrated that
treatment with anti-MIP-1 attenuated proteinuria but not the
accompanying neutrophil influx (16).
Mesangial cells are part of the renal glomerulus. They control the rate
of glomerular filtration and provide support for the capillary loops
(17). Mesangial cells are also important during glomerular
injury; they are positioned to receive proinflammatory signals from
capillary endothelium and infiltrating leukocytes. It is well
established that IL-1, TNF-, and LPS activate the in vitro synthesis
of various chemokines, including MCP-1 and RANTES, in mesangial cells
(6, 18, 19). This report extends these observations,
demonstrating that treatment of mesangial cells with MIP-2 or KC
induces the expression of MCP-1 and RANTES as well as autoinduction of
MIP-2 and KC. This chemokine cascade may contribute to the spatial and
temporal characteristics of renal inflammation.
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Methods and Materials |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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We purchased 6- to 12-wk-old BALB/cJ mice of either sex or BALB that were deficient for CXC-chemokine receptor-2 (CXCR2) from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained according to the guidelines of the Committee on Animals of the Harvard Medical School and those prepared by the Committee on Care and use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (Department of Health and Human Services Publication, National Institutes of Health 85-23, revised 1985).
Reagents
The recombinant mouse chemokines MIP-2, KC, MIP-1, and
MIP-1ß were purchased from R&D Systems (Minneapolis, MN). Eotaxin was
a gift of Millennium Pharmaceuticals, Inc. (Cambridge, MA). Because
some lots of chemokine contained traces of endotoxin, all chemokine
reagents were treated with Detoxi-Gel (Pierce, Rockford, IL) before use
to eliminate potential endotoxin contamination. Murine TNF- and
IFN- were procured from Genzyme (Cambridge, MA), whereas mouse
platelet-derived growth factor-BB (PDGF-BB) was purchased from Life
Technologies (Grand Island, NY). LPS and polymyxin B were obtained from
Sigma (St. Louis, MO). Mouse IL-1ß, 2H5 anti-MCP-1 mAb, and
biotinylated monoclonal 4E2 anti-MCP-1 ELISA reagent were purchased
from PharMingen (San Diego, CA). The 5F11 anti-MCP-1 Ab was
prepared as described elsewhere (20). Anti-KC antisera and
anti-KC mAb were obtained from R&D Systems. BSA was purchased from
United States Biochemicals (Cleveland, OH).
Mesangial cell isolation
Mesangial cells were obtained by outgrowth from mouse glomeruli obtained from four to ten mice. The method of Kreisberg et al. (21) was used for mesangial cell isolation. Briefly, kidneys were coarsely minced with scissors; tissue fragments were passed through a no. 60 mesh sieve (Curtin Matheson Scientific, Houston, TX) with a sterile rubber stopper and rinsed intermittently with 2% FCS/HBSS. The suspension was then sequentially passed through no. 100 and no. 200 sieves. Glomeruli were digested with 0.1% collagenase type IV (Sigma) and 0.1% trypsin (Life Technologies) for 30 min at 37°C before plating in 6-well tissue culture plates. Mesangial cells were cultured in DMEM with 20% heat-inactivated FCS in a 37°C humidified 10% CO2 incubator. Cellular outgrowth was observed 10–14 days after seeding. Mesangial cultures were fed biweekly and transferred at confluence. Cells were passaged in D-valine-substituted media to eliminate fibroblasts. After five passages the cells were apparently homogeneous as assessed by phase contrast and light microscopy and by their staining characteristics with anti-muscle actin Ab. Contamination with monocytes was excluded by the absence of reactivity with anti-Mac-1 (M1/70, Boehringer Mannheim, Indianapolis, IN) and anti-Ia mAb (34-5-3, a gift from Dr. S. Abromson-Leeman, Harvard Medical School, Boston, MA) as assessed by direct and indirect immunofluorescence, respectively. Confluent mesangial cell cultures were washed with serum-free medium before the addition of chemokines or cytokines, which were added in serum-free medium.
Culture of renal cortical epithelial cells
Renal cortical cells were isolated with minor modifications of an established technique (22). Briefly, eight mouse kidneys were coarsely minced with scissors and tissue fragments were sequentially passed through no. 60, no. 100, and no. 200 sieves. The cell suspension was incubated with a mixture of 0.1% collagenase and 0.1% trypsin for 15 min at 37°C. Dissociated cells were washed in HBSS and seeded into a 75-cm2 tissue culture flask. The cultures were maintained in DMEM supplemented with 10% FCS for 2–3 wk before use.
Collection of peritoneal exudate cells (PECs)
To induce leukocyte infiltration into the peritoneum, four to five mice were injected i.p. with 1 ml of 9% sodium casein (Sigma) in endotoxin-free PBS (23). After 16–24 h, animals were given a second casein injection. Three h later, peritoneal cells were harvested in 5–10 ml of HBSS without calcium chloride containing 0.5 mM EDTA. These cell populations contained 60–80% neutrophils, 20–30% macrophages, and <3% lymphocytes.
Detection of endotoxin
Limulus amebocyte lysate assays for the semiquantitation of endotoxin were conducted according to the manufacturer’s protocol (E-toxate, Sigma).
Capture ELISA for MCP-1 and KC quantitation
Immulon II 96-well microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 50 µl containing 10 µg/ml purified anti-MCP-1 mAb (5F11) or 2 µg/ml anti-KC mAb (48415) in 50 mM of sodium carbonate-bicarbonate buffer (pH 9) at room temperature for 5 h. After removing the excess capture Ab, the wells were filled with 200 µl of 3% BSA in PBS and incubated at 4°C overnight to saturate excess binding sites. After three washes with PBS, serial dilutions of the experimental samples diluted in 3% BSA/PBS were added to the plates for 3 h at 37°C. After three washes, 50 µl of biotinylated detector anti-MCP-1 mAb (4E2) or purified anti-KC antiserum in 3% BSA/PBS was added to the wells and incubated for 2 h at room temperature. After three additional washes, the plates were incubated with alkaline phosphatase-conjugated avidin for 2 h. After three final washes, plates were developed with p-nitrophenylphosphate (Sigma). Titrations of purified recombinant mouse MCP-1 (PharMingen) or KC (R&D Systems) were included in each experiment for the preparation of standardization curves (20).
Western blot
A total of 100 µl of culture supernatant was twice passed over a 50-µl heparin agarose column (Sigma) in a micropipet tip. The heparin beads were washed with 50 mM NaCl/10 mM HEPES (pH 7.2) buffer. The beads were then boiled for 5 min in 30 µl of sample buffer containing 5% 2-ME/2% SDS. Electrophoresis of a 20-µl protein sample was performed in 15% SDS-polyacrylamide minigels made with a MiniProtean II gel assembly kit (Bio-Rad, Hercules, CA) and electrophoresed at 150 V for 1.25–1.5 h. Proteins separated by SDS-PAGE were transferred to a 0.22-µm pore size nitrocellulose sheet (Bio-Rad). Sheets were blocked for 2 h at room temperature with 3% BSA/PBS and washed three times with PBS. Nitrocellulose sheets were incubated with 2H5 anti-MCP-1 mAb (10–20 µg/ml) in 3% BSA/PBS for 2 h at room temperature. The sheets were then washed three times for 10 min each with 3% BSA/PBS and reacted for 2 h at room temperature with a 1/1000 dilution of alkaline phosphatase-conjugated secondary Abs (Kirkegaard and Perry Laboratories, Gaithersburg, MD) in 3% BSA/PBS. The nitrocellulose sheet was finally washed three times for 5 min each with PBS; bound Abs were visualized by incubation with nitro blue tetrazolium/bromochloroindolyl phosphate (Kirkegaard and Perry Laboratories) at room temperature. The reaction was stopped by vigorous washing with PBS.
RNA isolation
RNA was isolated from cell suspensions according to the manufacturer’s protocol using an RNA Isolation Kit (Stratagene, La Jolla, CA). The RNA was further cleansed by precipitation followed by washing with isopropanol and 75% ethanol, respectively. RNA was finally suspended in 50 µl of diethyl pyrocarbonate-treated water.
RNase protection assay (RPA)
Assays for chemokine mRNA were conducted with multiprobe
templates according to the manufacturer’s protocol (RiboQuant assay
kit, PharMingen). The assay kits can simultaneously detect mRNA for
each of the following mouse chemokines: lymphotactin, RANTES, eotaxin,
MIP-1ß, MIP-1, MIP-2, IFN--inducible protein-10 (IP-10), MCP-1,
TCA3, and mRNA for the L32 and GAPDH housekeeping genes.
Chemotaxis assay
Cell migration was evaluated in 48-well Boyden microchambers separated with a 14-µm pore size polycarbonate filter (Neuroprobe, Cabin John, MD) as described previously (24). Mesangial cells (4 x 106/ml) were suspended in endotoxin-depleted DMEM with 1% BSA. A total of 50 µl of cells was added to the upper Boyden chamber, and 30 µl containing the indicated concentration of chemokine was added to the lower chamber.
RT-PCR
Single-stranded cDNA was synthesized from RNA using the
Superscript Preamplification System for first strand cDNA synthesis
(Life Technologies, Gaithersburg, MD) with the following modifications.
A total of 3 µg of total RNA was treated with 2 U of DNase-I (bovine
pancreas; Sigma) for 15 min at room temperature in an 18-µl volume
containing 1x PCR buffer and 2 mM MgCl2. Next,
it was inactivated by incubation with 2 µl of 25 mM EDTA at 65°C
for 10 min. Random hexamers (3 µl) were added and annealed to the RNA
according to the manufacturer’s protocol. The reverse transcription
reaction was performed according to protocol for a doubled reaction,
with the modification that 2.4 µl of 10x PCR buffer and 6.56 µl
MgCl2 were added. One-half of the reaction (19
µl) was removed for use as a control. This aliquot was not subjected
to reverse transcription. cDNA was then synthesized in a 20-µl
reaction containing 1.5 µg of total RNA as in the protocol, with a
parallel control reaction. The gene-specific PCR primers are listed in
Table I
. PCR was conducted in a 30-µl
reaction mixture with 0.6 µl of cDNA and 0.5 µl of each primer
under the manufacturer’s Taq DNA polymerase conditions
(Qiagen, Valencia, CA). The PCR program included preincubation at
94°C for 2 min, amplification for 27–30 cycles of PCR at 94°C for
45 s plus 55–58°C annealing for 45 s plus 72°C extension
for 45 s, and a final 72°C 3-min extension. A total of 6 µl of
the PCR mixtures was visualized on 3% agarose minigels. 
X174 RF
DNA/HaeIII fragments (Life Technologies) were included as
m.w. standards.
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Results |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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ELISA methods were used to measure MCP-1 levels in the
supernatants of mesangial cultures. The background level of MCP-1 in
unstimulated mesangial cells was consistently <1 ng/ml. In general,
detectable levels of MCP-1 were first identified in the serum
free-culture medium following a 42-h incubation with 
10 ng/ml MIP-2
or KC (Fig. 1A). These stimuli
did not induce synthesis of all cytokines, because the same
supernatants did not contain detectable levels of IL-1ß (data not
shown). Stimulation of mesangial cells with 100 ng/ml MIP-1 or 1000
ng/ml eotaxin failed to induce MCP-1 synthesis (Fig. 1A). To
establish that the Ag detected by ELISA represented conventional MCP-1
molecules, supernatants from unstimulated and LPS-, MIP-2-, or
eotaxin-treated cultures were adsorbed to heparin beads that were
subjected to Western blot analysis. The molecules detected in the
MIP-2- and LPS-treated culture supernatants were the same size as the
glycosylated recombinant mouse MCP-1 standard (Fig. 1B).
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A). Similar results were
obtained in RPAs (data not shown). In contrast, heat treatment (100°C
for 30 min) abrogated the capacity of MIP-2 and KC but not LPS to
stimulate MCP-1 synthesis (Fig. 2A). Finally, an anti-KC
mAb completely blocked KC-induced MCP-1 production (Fig. 2B). The failure of the anti-KC reagent to inhibit
IL-1ß-induced MCP-1 synthesis demonstrated the specificity of Ab
blocking (Fig. 2B).
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The dose response and kinetics of MIP-2- and KC-induced MCP-1
expression were examined at the RNA level using RPAs. The RNase
protection data were consistent with the protein data. A total of
25–50 ng/ml MIP-2 or KC were sufficient to stimulate MCP-1 mRNA
synthesis after 15 h (Fig. 3A). RNA synthesis was
detected after 3 h, but 24 h were required for peak RNA
expression (Fig. 3B). MIP-2 and KC also stimulated the
synthesis of RANTES and MIP-2 RNA (Fig. 3). Upon further exposure of
the autoradiographs, MIP-1, MIP-1ß, and occasionally IP-10 mRNA
were detected (Fig. 3C). When mesangial cells were
stimulated with 10 ng/ml IL-1ß or TNF-, mRNA for MCP-1 and RANTES
was readily induced (Fig. 3C). In addition, treatment with
IL-1ß and TNF- induced MIP-2 mRNA. IFN- also induced MCP-1
mRNA, but mRNA for other chemokines was not detected. Although PDGF
stimulates mesangial cell proliferation (20, 26, 27), it
did not induce significant transcription of chemokine RNA. As a
control, treatment with KC stimulated the production of mRNA for MCP-1,
RANTES, MIP-2, MIP-1, MIP-1ß, and IP-10 (Fig. 3C). In
contrast, RNA for TCA3, lymphotactin, and eotaxin was not detected
following the stimulation of mesangial cells with any of the indicated
stimuli (data not shown).
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A). To
identify KC protein, mesangial cells were incubated with various
stimulae for 24 h; afterward, the stimuli were washed out and the
cells were plated in serum-free media for an additional 48 h.
ELISAs were used to quantitate KC protein expression. After removal of
the original stimulus of 10 or 100 ng/ml KC, we detected autocrine
synthesis of 7 and 38 ng/ml, respectively, of KC protein. The levels of
autocrine KC synthesis are comparable with the levels of KC produced
following stimulation with MIP-2 or IL-1ß (Fig. 4B).
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To determine whether the mesangial cell responses to MIP-2 and KC
were cell type-specific, renal cortical epithelial cells were treated
with 1000 ng/ml KC, 200 ng/ml MIP-2, 10 ng/ml IL-1ß, or 10 µg/ml
LPS. The results shown in Fig. 5
demonstrate that little or no MCP-1 mRNA was detected following
stimulation with these high concentrations of KC or MIP-2. In contrast,
LPS treatment strongly stimulated MCP-1 as well as RANTES, MIP-1ß,
MIP-1, MIP-2, and IP-10 RNA synthesis. MCP-1 mRNA was also induced
following treatment with IL-1ß.
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. PECs produced MCP-1 following stimulation with LPS, IL-lß,
or TNF-. In addition, neutrophil chemotaxis was demonstrated
following KC or MIP-2 stimulation (data not shown). In contrast, these
cells failed to make detectable levels of MCP-1 protein following MIP-2
or KC stimulation (Fig. 6).
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The most prominent feature of chemokines is their ability to
induce cell migration. To examine the chemotactic capacity of MIP-2 or
KC, mesangial cells were placed in a Boyden microchamber with varying
doses of MIP-2 or KC and the positive and negative control chemokines
TCA3 and MIP-1ß, respectively (24). MIP-2-induced
migratory responses displayed a bell-shaped dose-response curve
characteristic of chemokines (24, 28). Peak chemotactic
responses were noted with 10 ng/ml MIP-2 and 10–100 ng/ml KC. As noted
elsewhere, TCA3 was a less efficacious chemoattractant; a total of
100-1000 ng/ml TCA3 were required to stimulate optimal mesangial cell
migration. As expected (24), MIP-1ß failed to attract
mesangial cells at all concentrations tested (Fig. 7).
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Chemokine receptor expression on mesangial cells
Mouse leukocytes bind MIP-2 and KC through a common
seven-transmembrane-spanning G protein-coupled receptor, CXCR2
(29, 30). We examined primary mesangial cells for
expression of CXCR2, CXCR3, and CXCR4 by RT-PCR. Although CXCR2, CXCR3,
and CXCR4 products were readily detected in control PECs, we were
unable to amplify these products from mesangial cells under identical
experimental conditions (Fig. 8A).
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B) and protein (Table II) following MIP-2 or KC stimulation. In
addition, RANTES and MIP-2 RNA were also detected following KC or MIP-2
stimulation of CXCR2-deficient mesangial cells. (Fig. 8B)
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Discussion |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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mesangial cells release chemokines and other
proinflammatory mediators (18, 33, 34). This report
demonstrates an additional pathway for chemokine induction. Mesangial
cells display a unique amplification mechanism in which the
CXC-chemokines MIP-2 or KC readily induce MCP-1 and RANTES expression;
mRNA for MIP-1, MIP-1ß, and IP-10 was induced with lower
efficiency. Interestingly, MIP-2 and KC display autocrine activity,
stimulating the production of additional MIP-2 and KC. Similar
properties were reported previously for the human CXC-chemokine
melanoma growth-stimulatory activity factor/gro-, which stimulated
autoinduction in HUVEC (35). The autoinduction of
chemokines by mesangial cells suggests a mechanism for perpetuating
inflammatory responses within the parenchyma.
The murine chemokines KC and MIP-2 share structural homology with each
other (29, 36) and with the human chemokines melanoma
growth-stimulatory activity factor (also termed gro-), gro-ß, and
gro- (36, 37). Due to the profusion of human
CXC-chemokines with redundant structural and functional activities,
more specific homology assignments are not possible (36).
KC and MIP-2 generally induced similar patterns of chemokine
transcription in mesangial cells.
Only a limited number of reports have described the effects of
chemokines on mesangial cells. Barnes et al. (38)
demonstrated that micromolar concentrations of the human CXC-chemokine
platelet factor-4 inhibited mesangial cell proliferation. Another
report indicated that nanomolar concentrations of human IL-8 stimulated
the adhesion of mesangial cells to fibronectin (24). In
the current report, induction of migratory responses and protein
synthesis was also achieved with nanomolar concentrations of MIP-2 or
KC. Similarly, low concentrations of the CC-chemokine TCA4 are known to
induce mesangial cell migration (39), but 10-fold higher
concentrations of TCA3 are required to stimulate chemotaxis (Fig. 7).
The affinity range of conventional chemokine receptors is in the
nanomolar range, suggesting the physiologic relevance of the MIP-2 and
KC interactions (29, 30, 40, 41).
Although it is clear that MIP-2 and KC can act on primary mesangial
cells, the receptor(s) responsible for transmitting the MIP-2/KC
signals remains unknown. CXCR2 is the common leukocyte receptor for
MIP-2 and KC, binding these ligands with high affinity (29, 30, 41). We were unable to detect CXCR2 on mesangial cells by
RT-PCR, and mesangial cells from CXCR2-deficient mice were responsive
to MIP-2 and KC, indicating that additional chemokine receptors for
these ligands remain to be discovered. The finding that a neutralizing
anti-KC mAb that inhibits neutrophil chemotaxis also blocks
mesangial cell responses (Fig. 2B) suggests that the same KC
epitope is involved in binding to both CXCR2 and the unidentified
mesangial cell receptor.
The ability of MIP-2 and KC to trigger mesangial cell chemokine induction seems to be tissue-specific, because similar effects were not noted on renal cortical epithelial cells or peritoneal neutrophils and macrophages. However, analysis of additional smooth muscle-like tissues should be undertaken. In summary, these data suggest that MIP-2 and KC can stimulate a chemokine amplification cascade that may perpetuate glomerular inflammation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martin E. Dorf, Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115.
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; IP-10, IFN--inducible protein-10; PDGF, platelet-derived growth factor; RPA, RNase protection assay; CXCR, CXC-chemokine receptor; PEC, peritoneal exudate cell.
Received for publication March 1, 1999. Accepted for publication July 20, 1999.
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References |
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TopAbstractIntroductionMethods and MaterialsResultsDiscussionReferences |
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and MIP-1ß. Science 260:355. induce gene expression and production of leukocyte chemotactic factors, colony-stimulating factors, and interleukin-6 in human mesangial cells. Am. J. Pathol. 138:991.[Abstract]
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), KC (•),
MIP-1
), or eotaxin (
) in serum-free medium. Supernatants
were collected after 48 h and assayed for MCP-1 protein by ELISA.
B, Immunoblot of mesangial cell supernatants. Mesangial
cell preparations were cultured for 42 h with medium (lane
1), 1000 ng/ml LPS (lane 2), 100 ng/ml MIP-2
(lane 3), or 1000 ng/ml eotaxin (lane 4).
A total of 100 µl of each conditioned medium was adsorbed onto
heparin beads. The eluted proteins were run on a 15% SDS-PAGE gel and
transferred. The blot was probed with anti-MCP-1. An aliquot of
Chinese hamster ovary cell-derived mouse recombinant MCP-1 (lane
5) was included as a standard.
), and MIP-1ß (