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*
Department of Pathology, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Department of Cell Physiology, University of Nijmegen, The
Netherlands
Diaclone, Besançon, France
§
Renal Division, Emory University School of Medicine and Veterans Affair
Medical Center, Atlanta, Georgia.
Correspondence to Dr. José G. van den Berg, Department of Pathology, L2-256, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Phone: +31 20 566 4935; Fax: +31 20 696 0389; E-mail: j.g.vandenberg{at}amc.uva.nl
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-13R1, and
IL-13R2. Protein expression of IL-4R and IL-13R2 by GVEC
in human kidney biopsies and by cultured human GVEC was detected by
immunohistochemistry. Western blotting demonstrated phosphorylation of STAT6
in cultured GVEC upon incubation with IL-4 or IL-13. This indicated signal
transduction via the heterodimeric receptor complex IL-4R2, which is composed
of the IL-4R and the IL-13R1. Direct effects on GVEC function
were examined in monolayer experiments. IL-4 and IL-13 dose-dependently
decreased transepithelial electrical resistance of monolayers of rat GVEC to
approximately 30 and 40% of baseline values, respectively. The transepithelial
electrical resistance decrease was associated with a significant increase in
short-circuit current, whereas no changes were observed in the transmonolayer
flux of the macromolecules horseradish peroxidase (molecular weight, 44 kD)
and 14C-mannitol (molecular weight, 182 Da). No changes in cell
structure were observed with electron microscopy. It is concluded that by
binding to specific IL-4/IL-13 receptors, IL-4 and IL-13 can exert specific
effects on GVEC function, which could be of pathogenetic relevance for
glomerular injury in MCN. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MCN can be treated successfully with immunosuppressive drugs, and the involvement of immunologic processes in the pathogenesis of MCN has been suggested (1). Several groups have described a factor with permeability-enhancing properties in the supernatant of stimulated peripheral blood mononuclear cells from patients with MCN. After infusion, this supernatant induced proteinuria and foot process retraction in rats (2,3,4). Other investigators have focused on the cytokine profile of T lymphocytes of patients with MCN. Recently, it was shown that in patients with MCN T lymphocytes produce more interleukin-13 (IL-13) than healthy control subjects and patients in remission (5, 6).
IL-13 is a pleiotropic cytokine that, like IL-4, is produced by
appropriately stimulated CD4+ T-helper2 (Th2) lymphocytes. Activation of Th2
lymphocytes plays a key role in IgE-mediated inflammation, as in atopic
syndromes and in immune responses to parasites and fungi. A possible role for
Th2 cytokines in the induction of proteinuria in MCN is supported by clinical
observations, such as the association of MCN with atopy, and the apparent
induction of MCN by allergic events in some patients
(7). In earlier studies, we
showed that IL-4 can exert specific effects on rat GVEC in vitro
(8). IL-4 and IL-13 have
overlapping effector profiles
(9). This overlap is at least
partially due to the shared use of a heterodimeric IL-4/IL-13 receptor
complex, the IL-4R2. This receptor complex is composed of the IL-4 binding
IL-4R chain and the IL-13 binding IL-13R1 chain. Alternatively,
the IL-4R chain can be associated with the common 
-chain
(c-chain) to form the heterodimeric IL-4R1, which signals only after
binding of IL-4 (10). Another
IL-13-binding chain, the IL-13R2 chain, is believed to bind IL-13 with
high affinity without subsequent signaling
(11). There is now growing
evidence that in asthma IL-13 functions as a key mediator independently of
IL-4 (12,
13). Correspondingly, it was
shown that in MCN, T lymphocytes are prone to produce IL-13 rather than IL-4
(5,
6).
Whether the observed increased production of IL-13 by T lymphocytes of patients with MCN plays a role in the pathogenesis of this disease is as yet unclear. We hypothesized that IL-13 may play an important role in the development of proteinuria, by exerting direct effects on GVEC. To address this hypothesis, we studied effector functions of IL-4 and IL-13 on rat GVEC in vitro. Furthermore, we examined the expression of IL-4 and IL-13 receptors on human and rat GVEC, both in vitro and in vivo. We show that IL-4 and IL-13 act directly on GVEC in vitro, probably by binding to a functional IL-4R2, which is also likely to be expressed by GVEC in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(MAB230, mouse IgG2a) was purchased
from R&D Systems (Abingdon, United Kingdom). Monoclonal antibody to human
IL-13R2 (B-D13, mouse IgG1) was described previously
(16). Polyclonal antibody to
phosphorylated STAT6 (Phospho-Stat6 [Tyr641], rabbit IgG) was purchased from
New England Biolabs (Beverly, MA). Monoclonal antibody to STAT6 (SC-1689,
mouse IgG1) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cells
Established lines of rat GVEC
(17), human GVEC
(18), and rat mesangial cells
(19) were used. Cells were
cultured at 37°C in plastic flasks in a humidified 5% CO2/95%
air incubator. Rat GVEC were grown on a type I collagen gel (Vitrogen 100;
Collagen Corp., Palo Alto, CA); 0.2% collagenase type IA (330 U/mg; Sigma, St.
Louis, MO) in Hanks' balanced salt solution (Life Technologies, Breda, The
Netherlands) was used to detach cells before each passage. Human GVEC and rat
mesangial cells were grown on plastic; trypsin (DIFCO Laboratories, Detroit,
MI) was used to detach cells. The GVEC culture medium contained 45% Dulbecco's
modified Eagle's medium (ICN, Costa Mesa, CA), 45% HAM-F10 (Life
Technologies), 1% K1 hormone mix (5 µg/ml insulin, 25 ng/ml prostaglandin
E1, 0.5 nM T3, 10 nM sodium selenite, 5 µg/ml transferrin, 50 nM
hydrocortisone [all ingredients from Sigma] in Hanks' balanced salt solution),
1% insulin (Sigma), and 5% Nu serum (Becton Dickinson, Bedford, MA). Mesangial
cell culture medium was composed of 95% Dulbecco's modified Eagle's medium and
5% fetal calf serum (Life Technologies). Culture media were supplemented with
100 U/ml penicillin and 100 µg/ml streptomycin (both from Life
Technologies).
Isolation of Glomeruli
To obtain human glomerular tissue, kidneys were used that had been resected
because of chronic renal allograft rejection (n = 2), acute renal
allograft rejection (n = 1), or recurrent pyelonephritis (n
= 1). From each kidney, 20 glomeruli were microdissected from a 10-µm-thick
cryostat section with a Positioning Ablation Laser Microscope (P.A.L.M.
Mikrolaser Technologie, Bernried, Germany). Rat glomeruli were isolated under
sterile and RNase-free conditions from normal Brown Norway rats by
differential sieving and centrifugation of minced kidney cortices.
cDNA Synthesis
Total RNA was isolated from cultured human and rat GVEC, from cultured rat
mesangial cells, and from isolated rat glomeruli, using Trizol Reagent (Life
Technologies) according to the manufacturer's instructions. From isolated
human glomerular tissue, cDNA was synthesized without prior RNA isolation. For
cDNA synthesis, either 10 µg of isolated RNA or the isolated human
glomerular tissue was incubated with 5 nmol of Pd(N)6 primer (Pharmacia
Biotech, Roosendaal, The Netherlands) for 10 min at 65°C. After cooling on
ice, the reverse transcription (RT)-reaction mixture was added to a final
volume of 50 µl, containing 400 U of Moloney murine leukemia virus reverse
transcriptase (Life Technologies), 8 mmol/L dithiothreitol, 1 mmol/L of each
dNTP, 1x first-strand buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl,
3 mmol/L MgCl2), and 60 U of RNase inhibitor (Boehringer Mannheim,
Almere, The Netherlands). The reaction was performed for 1 h at 37°C.
Subsequently, the reverse transcriptase was inactivated by heating the sample
for 10 min at 95°C.
Polymerase Chain Reactions
Species-specific primers for synaptopodin, c-chain, IL-4R,
IL-13R1, and IL-13R2 were designed on the basis of sequences
available from the Entrez sequence databank from the National Center for
Biotechnology Information and synthesized by Pharmacia Biotech. The sequences
of the primers are listed in Table
1. In each PCR, 2 µl of the cDNA reaction mixture was used or,
for nested PCR, 5 µl of mixture containing the initial PCR product. Water
was used as a control to exclude contamination. The PCR mixture contained
1x Taq buffer (20 mmol/L Tris-HCl, 50 mmol/L KCl, pH 8.4), 0.2
mmol/L of each dNTP, 2.5 mmol/L MgCl2, 2 U of Taq
polymerase (Life Technologies), and 0.5 µmol/L of each primer. Thirty
cycles of amplification, or 35 cycles for nested PCR, were performed in the
thermal cycler (PTC-100; M. J. Research, Inc., Watertown, MA), i.e.,
successively for 60 s at 95°C, 60 s at 58°C, and 60 s at 72°C. The
reaction was completed during 7 min at 72°C. PCR products were analyzed in
10-µl samples on a 1.5% agarose gel (Sigma) containing ethidium
bromide.
|
Amplification of contaminating genomic DNA during the RT-PCR procedures was excluded by PCR on isolated genomic human and rat DNA, which did not yield transcripts of the size expected for the target mRNA.
Sequencing of PCR Products
PCR products were isolated from the PCR mixture with a QIA-Quick kit
(Qiagen, Hilden, Germany) and were sequenced using an ABI sequencer (Perkin
Elmer Corp., Norwalk, CT) with a dyeterminator cycle-sequencing kit (Perkin
Elmer) according to the manufacturer's instructions.
Immunohistochemistry
The protein expression of IL-4R and IL-13R2 was analyzed by
immunohistochemistry on cryostat sections of human renal tissue and on
cultured human GVEC. The human renal tissue was derived from histologically
normal parts of kidneys that had been resected because of a Grawitz tumor
(n = 3) and from 33 renal biopsy specimens from patients with the
following diagnoses: MCN (n = 8), chronic renal allograft rejection
(n = 3), acute renal allograft rejection (n = 3), membranous
glomerulopathy (n = 3), diabetes mellitus nephropathy (n =
3), focal segmental glomerulosclerosis (n = 3), IgA nephropathy
(n = 4), tubulointerstitial nephritis (n = 4), and Wegener's
vasculitis (n = 2).
Nonfixed, 4-µm-thick sections of human renal tissue and acetone-fixed human GVEC, which had been cultured on glass slides, were preincubated with 10% normal goat serum (Sera Lab, Sussex, United Kingdom), followed by incubation with the primary antibodies overnight at 4°C. Appropriate dilutions in phosphate-buffered saline (PBS) were determined for each antibody. Endogenous peroxidase activity was blocked by incubation with 0.1% NaN3 and 0.03% H2O2 (both from Sigma) in PBS. Subsequently, antibody binding was detected with isotype-specific horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig antibodies (Southern Biotechnology Associates, Birmingham, AL); 10% normal human ABO-serum (CLB, Amsterdam, The Netherlands) was added to the secondary antibody to inhibit possible cross-reactivity to human immunoglobulins. Peroxidase activity was detected with 3-amino-9-ethylcarbozole (Sigma) and 0.03% H2O2. Negative controls were performed by replacing the first-step antibody by incubation buffer only or by isotype-matched and species-matched monoclonal antibodies that do not bind to human tissue (Dako).
Monolayer Experiments
Because the rat GVEC line forms continuous monolayers, whereas the human
GVEC line does not, we used rat GVEC for monolayer experiments. Cells were
seeded on Transwell porous filters with a pore diameter of 0.4 µm and a
growth area of 0.33 cm2 (Costar, Badhoevedorp, The Netherlands) at
a density of 150,000 cells/cm2. The inner and outer chambers
contained 0.25 and 1.2 ml of medium, respectively. For most experiments, the
cells were seeded in the inner chamber, on the top of the filter. In this
situation, the inner and outer chambers represented the apical and basolateral
compartments, respectively. In some experiments, cells were grown on the lower
side of the filter instead, and the inner and outer chambers represented the
basolateral and the apical compartments, respectively. Cytokines were added to
the apical compartment, to the basolateral compartment, or to both
compartments.
Transepithelial electrical resistance (TER) was measured with a Millipore
apparatus. After reaching TER values >2.5 k
xcm2,
the confluent monolayers were treated as detailed below. For short-circuit
current (ISC) measurements, confluent GVEC monolayers were mounted
in an Ussing chamber and bathed at 37°C with incubation medium containing
(in µM): 140 NaCl, 2 KCl, 1 K2HPO4, 1
KH2PO4, 1 MgCl2, 1 CaCl2, 5
glucose, 5 L-alanine, and 10 Hepes-Tris, pH 7.4. The solutions bathing the
monolayers were connected via agar bridges and Ag-AgCl electrodes to a
voltage-clamp current amplifier (Physiological Instruments, San Diego, CA);
the ISC was recorded and was used as an estimate of transcellular
ion transport.
To measure the transmonolayer flux of macromolecules, in some experiments 14C-mannitol (0.25 µCi/ml) or HRP (10 µg/ml) (both from Sigma) was added to the medium in the basolateral chamber, 2 d after addition of the cytokines to the basolateral chamber. Subsequently, 14C-mannitol radioactivity or HRP enzyme activity, respectively, was measured in both chambers. 14C-mannitol radioactivity was determined by liquid scintillation. HRP activity was determined using orthophenyl-diamine (Eastman Kodak Co., Rochester, NY) and H2O2 as substrates; the absorbance at 492 nm was quantified by a Bio-Rad microplate reader.
To measure cell death, the lactate dehydrogenase (LDH) release from the GVEC monolayers was determined. Three days after incubation with the cytokines, samples were taken from both the apical and the basolateral chambers, and from the cells after detergent extraction in a total volume of 150 µl with 2% Triton X-100. LDH was measured by spectrophotometry on a Hitachi 747 (Hitachi, Tokyo, Japan) at 37°C with lactate as the substrate.
To study the ultrastructure of the rat GVEC monolayers, GVEC cultured on the Transwell filters were fixed with 2.5% glutaraldehyde. Filters and cells were cut and embedded in Epon (LADD Research Industries, Inc., Williston, VT). The integrity of the monolayers was studied by electron microscopy of ultrathin sections, with special attention to cell-cell and cell-filter adhesion. Monolayers that had been basolaterally exposed to IL-4 or to IL-13 for 3 d were compared with untreated monolayers of the same age.
Western Blot Analysis of STAT6 Phosphorylation
Phosphorylation of STAT6 on the Tyr641 residue in cultured human and rat
GVEC was determined by Western blotting. Human GVEC grown on plastic were
incubated with human IL-4 or IL-13 (both 100 ng/ml). Rat GVEC were seeded onto
large Transwell porous filters (pore diameter, 0.4 µm; growth area, 4.7
cm2; Costar). The apical and basolateral compartments contained 2.0
and 3.0 ml of culture medium, respectively. When TER values of approximately 4
kxcm2 were reached, GVEC monolayers were incubated
with rat IL-4 (10 ng/ml) or rat IL-13 (1 U/ml) in the basolateral
compartments.
After incubation with the indicated cytokines for 0, 10, 20, 30, or 60 min, cells were lysed with sample buffer, containing 100 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.001% bromphenol blue, and 100 mM 2-mercaptoethanol. Proteins in cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis and were blotted onto nitrocellulose paper. Staining with Ponceau S confirmed effective protein transfer. Blocking was carried out with 5% milk powder (Nutricia, Zoetermeer, The Netherlands) in Tris-buffered saline containing 0.1% Tween 20 (Sigma) (TBSTM) for 1 h at room temperature. The nitrocellulose membranes were then incubated overnight at 4°C with rabbit anti-Phospho-STAT6 antibody; incubation with PBS was used as a negative control. After extensive washing with TBSTM, the membranes were incubated with HRP-conjugated mouse anti-rabbit IgG (Dako) for 1 h. Binding was visualized using a chemiluminescence detection system (ECL; Amersham, Buckinghamshire, United Kingdom) and analyzed with a Lumi-Imager F1 Workstation (Roche Molecular Biochemicals, Basel, Switzerland).
Subsequently, to remove primary and secondary antibodies, membranes were incubated in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) at 50°C for 30 min. After washing and blocking in TBSTM for 1 h at room temperature, membranes were reprobed with anti-STAT6 antibody. Immunodetection was performed as described above, except that HRP-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates) was used as the secondary antibody.
Statistical Analyses
Values are expressed as means ± SD when appropriate. One-way ANOVA
tests were performed to analyze differences in ISC, in
transmonolayer flux of 14C-mannitol or HRP, and in LDH release
between control cultures and cultures incubated with IL-4 or IL-13.
Differences were considered statistically significant at P <
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-13R1, and
IL-13R2 were detected by RT-PCR in the isolated human glomeruli of all
patients studied, in isolated rat glomeruli, in both human and rat GVEC lines,
and in rat mesangial cells. Messenger RNA for the c-chain was detected
only in isolated rat glomeruli and in rat mesangial cells
(Figure 1). In isolated human
and rat glomeruli and in both GVEC lines, expression of mRNA for synaptopodin
was shown. In addition to identification of the RT-PCR products by their
expected size, unequivocal proof of the authenticity of the amplicons was
obtained by sequencing, which yielded results identical to the published
sequences.
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The expression of the IL-4R chain and the IL-13R2 chain on
human GVEC was further demonstrated at the protein level by
immunohistochemistry. In human renal biopsies from patients with various
kidney diseases (see Materials and Methods), we found glomerular staining with
the respective monoclonal antibodies to these receptor chains, as shown in
Figure 2. GVEC expressed both
IL-4R and IL-13R2 in all cases studied, whereas expression of
these proteins by mesangial cells and by glomerular endothelium was variable.
This limited study did not reveal typical staining patterns that could be
correlated to the diagnostic groups. Anti-IL-4R as well as
anti-IL-13R2 also specifically bound to human GVEC cultured on glass
slides (not shown).
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STAT6 Phosphorylation
Because binding of the IL-4R2 both by IL-4 and by IL-13 is known to be able
to induce signal transduction via STAT6
(20), we examined STAT6
phosphorylation in cultured rat and human GVEC by Western blotting with an
antibody specific for an epitope containing the phosphorylated Tyr641 residue
of STAT6. In untreated cells, no STAT6 phosphorylated at Tyr641 was detectable
(Figure 3). In human GVEC,
phosphorylated STAT6 was detected as a protein of approximately 100 kD within
10 min of incubation with IL-4 (Figure
3) and was also detected at 30 and 60 min of incubation with IL-4
(not shown). In rat GVEC cultured on porous filters, phosphorylated STAT6 was
only detected after 30 and 60 min of incubation with IL-4
(Figure 3). Upon incubation
with IL-13, Tyr641 phosphorylation of STAT6 was detected in rat GVEC, but not
in human GVEC. Reprobing the membranes with an antibody against STAT6 revealed
that total STAT6 was equally present in both treated and untreated samples.
After incubation with IL-4 or IL-13, an additional protein (with an apparent
molecular weight of approximately 85 kD) was detected in rat GVEC with the
antibody against phosphorylated STAT6, but not with the antibody against total
STAT6 (Figure 3).
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Transepithelial Electrical Resistance
Within 3 d from seeding onto filters, rat GVEC formed confluent monolayers
with TER values of 2.5 kxcm2, rising to a maximum of
6.0 kxcm2 after 5 d. IL-4 and IL-13, when added to the
basolateral compartment of the cell monolayer at day 0, caused a strong
decrease in TER (Figure 4).
After addition of these cytokines, the TER dropped within 1 d and was
maximally decreased after 2 to 3 d, reaching values of 1.0 to 1.5
kxcm2. Administration of the cytokines to the apical
compartment of the Transwell filters did not affect TER
(Figure 5), regardless of
whether the apical compartment was represented by the inner chamber or by the
outer chamber, indicating that this specificity was not due to differences in
volumina (not shown). The effect of IL-4 and IL-13 was dose-dependent
and could be specifically blocked by OX81 (monoclonal anti-IL-4 antibodies,
0.1 ng/ml) and by anti-IL-13 (rabbit polyclonal antibodies, 1/100 dilution),
respectively, but not by species-matched and isotype-matched nonspecific
antibodies in comparable dilutions (Figure
6).
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Short-Circuit Current (ISC)
Transcellular ion transport across GVEC monolayers was measured as
ISC. After incubation of the monolayers with IL-4 or IL-13 for 3 d,
the ISC was significantly increased
(Figure 7).
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Monolayer Permeability to Macromolecules
To assess whether the decrease in TER induced by IL-4 and IL-13 was
paralleled by an increase in monolayer permeability to macromolecules, we
quantified the transmonolayer flux of two organic compounds of different
molecular size. 14C-Mannitol (molecular weight, 182 Da) and HRP
(molecular weight, 44 kD) were added to the basolateral compartments of
monolayers that had been pretreated with cytokines for 2 d. Subsequently, the
appearance of these compounds in the apical compartment was measured after 24
and 48 h. As shown in Table 2,
pretreatment of the GVEC with IL-4 or IL-13 did not increase monolayer
permeability to these macromolecules.
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Electron Microscopic Findings
The ultrastructure of rat GVEC monolayers was studied by electron
microscopy. No obvious structural differences were observed between control
monolayers and monolayers that had been treated with IL-4 or IL-13 for 3
d.
Cytotoxicity
LDH activity in the apical supernatant was not significantly different for
GVEC cultures incubated with cytokine for 3 d compared to controls of the same
age (266 ± 63 U/L after IL-4, 224 ± 19 U/L after IL-13, and 224
± 43 U/L in controls, n = 8). In the basolateral medium, LDH
activity was negligible and not different among the groups. Intracellular LDH
content in the cytokine-treated cells was significantly higher than in
controls (751 ± 97 U/L after IL-4, 726 ± 121 U/L after IL-13,
and 248 ± 87 U/L in controls, P < 0.001). Taken together,
the release of LDH into the supernatants from cytokine-treated cells was
similar to that from controls, indicating that the decrease in TER induced by
IL-4 and IL-13 was not due to cytotoxicity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(49) in
peripheral blood mononuclear cells from patients with MCN. To circumvent
artifacts induced by stimulation with mitogens, more recently the unstimulated
production of cytokines by T lymphocytes of patients with MCN was studied, and
increased expression and production of IL-13 were found
(5,6).
These investigators suggested that circulating IL-13 may act on monocytes by
triggering the production of vascular permeability factor, which in turn would
increase glomerular permeability
(6). We hypothesized that IL-4
and IL-13 may directly act on GVEC. To address this hypothesis, we first
studied the expression of the specific IL-4/IL-13 receptors on GVEC. At the
mRNA level, we showed that rat and human GVEC express the two components of
the IL-4R2, as well as the IL-13R2. In isolated rat and human
glomeruli, the same pattern of expression was detected. Only in rat glomeruli
did we find expression of mRNA for the c-chain. This mRNA is probably
derived from passenger cells or from mesangial cells rather than from GVEC,
since cultured rat mesangial cells expressed c-chain mRNA. At the
protein level, immunohistochemistry revealed expression of IL-4R and
IL-13R2 by GVEC in human glomeruli and by human GVEC in culture.
Because no specific antibodies to IL-13R1 are as yet available, we
could not study protein expression of this receptor chain.
IL-4 and IL-13 are known to activate STAT6 by binding to the IL-4R2 in
human colon carcinoma cell lines
(20) and in human umbilical
vascular endothelial cells
(50). In rat GVEC in
vitro, we showed induction of STAT6 phosphorylation both by IL-4 and by
IL-13, indicating that these cells express a functional IL-4R2. In the human
GVEC line, STAT6 phosphorylation was only observed upon incubation with IL-4,
which, in the absence of the c-chain, is probably the result of binding
to a functional IL-4R2. The lack of STAT6 phosphorylation in cultured human
GVEC in the presence of IL-13 might be due to overexpression of the
high-affinity receptor IL-13R2, which does not signal after binding, as
has been described for cultured human synovial fibroblasts
(11).
Functioning of the IL-4R2 on rat GVEC was further evidenced by the induction of specific changes in cell function both by IL-4 and by IL-13. To study these direct effects of IL-4 and IL-13 on GVEC function, we measured TER over a monolayer of rat GVEC in a Transwell system. TER is a commonly used estimate of epithelial cell function, representing at least three characteristics of an epithelial monolayer: monolayer permeability, transcellular ion transport, and cell structure. We showed that IL-4 and IL-13 specifically decreased the TER of a monolayer of rat GVEC. This decrease was associated with an increase in ISC, suggesting an increase in transcellular ion transport, but it was not accompanied by an increased monolayer permeability to macromolecules, or by cell death. Electron microscopic examination of the GVEC monolayers revealed no gross structural changes induced by IL-4 or IL-13. Of course, TER, ISC, and monolayer permeability in vitro cannot be translated to glomerular permeability in vivo. Furthermore, these rat GVEC in culture do not form foot processes. Therefore, the relevance of our findings on GVEC function and on GVEC structure in vitro with respect to induction of proteinuria and foot process effacement warrants further investigation in models in vivo.
In conclusion, we show that IL-4 and IL-13 exerted specific effects on GVEC in vitro, most likely by binding to the IL-4R2, which may also be expressed by GVEC in vivo. In MCN, circulating IL-13 produced by triggered T lymphocytes (5,6) could directly act on GVEC by binding to the IL-4R2, and thus possibly play a role in the pathogenesis of the specific changes in GVEC structure and glomerular permeability observed in these patients.
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Acknowledgments |
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This work was supported by grants from the Dutch Kidney Foundation (No. C98.1720) and from The Netherlands Organization for Scientific Research (NWO, Grant 920-03-062). We thank Dr. J. D. Sraer (Hospital Tenon, Paris, France), Dr. D. J. Salant (Boston University Medical Center, Boston, MA), and Dr. D. Mason (University of Oxford, Oxford, United Kingdom) for providing cell lines, and Mrs. G. E. E. van Noppen for careful revision of the manuscript.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. B. Madden, L. Whitman, C. Sullivan, W. C. Gause, J. F. Urban Jr., I. M. Katona, F. D. Finkelman, and T. Shea-Donohue Role of STAT6 and Mast Cells in IL-4- and IL-13-Induced Alterations in Murine Intestinal Epithelial Cell Function J. Immunol., October 15, 2002; 169(8): 4417 - 4422. [Abstract] [Full Text] [PDF]
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 W. Debinski, B. H. Joshi, G. E. Plautz, and R. K. Puri Correspondence re: B. H. Joshi et al., Interleukin-13 Receptor {alpha} Chain: A Novel Tumor-associated Transmembrane Protein in Primary Explants of Human Malignant Gliomas. Cancer Res., 60: 1168-1172, 2000. Cancer Res., July 1, 2001; 61(14): 5660 - 5660. [Full Text] [PDF]
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 J. G. Van Den Berg, J. Aten, C. Annink, J. H. Ravesloot, E. Weber, and J. J. Weening Interleukin-4 and -13 promote basolateral secretion of H+ and cathepsin L by glomerular epithelial cells Am J Physiol Renal Physiol, January 1, 2002; 282(1): F26 - F33. [Abstract] [Full Text] [PDF]
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