1 Institute for Prevention of Cardiovascular Disease, Ludwig-Maximilians Universität München, D-80336 Munich, and 2 Department of Molecular Cardiovascular Research, University Hospital Aachen, D-52074 Aachen, Germany
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ABSTRACT |
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The activation of nuclear factor-B
(NF-
B) is required for the induction of many of the adhesion
molecules and chemokines involved in the inflammatory leukocyte
recruitment to the kidney. Here we studied the effects of NF-
B
inhibition on the machinery crucial for monocyte infiltration of the
glomerulus during inflammation. In mesangial cells (MC), the protease
inhibitors MG-132 and N-
-tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of I
B-
prevented the complete I
B-
degradation following tumor necrosis factor-
(TNF-
) stimulation. This resulted in a marked inhibition of
TNF-
-induced expression of mRNA and protein for the immunoglobulin
molecules intracellular adhesion molecule-1 and vascular cell adhesion
molecule-1 and the chemokines growth-related oncogene-
, monocyte
chemoattractant protein-1, interleukin-8, or fractalkine in MC.
Finally, the inhibition of I
B-
degradation or I
B-
overexpression suppressed the chemokine-induced transendothelial
monocyte chemotaxis toward MC and the chemokine-triggered firm adhesion
of monocytic cells to MC. The inhibition of NF-
B by pharmacological
intervention or gene transfer may present a multimodal approach to
control the machinery propagating inflammatory recruitment of monocytes
during glomerular disease.
N--tosyl-L-lysine chloromethyl ketone; MG-132; adenovirus; adhesion molecules; chemokines; nuclear factor-
B
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INTRODUCTION |
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MONOCYTE ADHESION AND
EMIGRATION involves sequential and overlapping interactions of
signal molecules and is fundamental to the inflammatory response.
Whereas monocyte rolling is mediated by selectins and their selectin
counterreceptors, firm adhesion crucially depends on the binding of
1- and
2-integrins to their endothelial
ligands vascular cell adhesion molecule-1 (VCAM-1) and intracellular
adhesion molecule-1 (ICAM-1) as well as immobilized chemokines.
Subsequent exposure to chemokine gradients may then be responsible for
transendothelial migration (3, 6, 9, 22, 28, 32, 36).
In various models of renal diseases, mesangial cells (MC) have been
implicated in glomerular leukocyte recruitment. MC are myofibroblast-like cells that, together with the extracellular matrix,
form the specialized pericapillary tissue in the glomerulus and thus
contribute to the regulation of ultrafiltration and microcirculation (7, 13, 26, 27). Various inflammatory stimuli have been demonstrated to increase the expression of the immunoglobulin gene
superfamily members ICAM-1 and VCAM-1 (7, 14, 21), CC-chemokines monocyte chemoattractant molecule-1 (MCP-1) and RANTES, and CXC-chemokines interleukin (IL)-8 and growth-related oncogene- (GRO-
) (27). We have recently shown that
GRO-
and IL-8, induced by tumor necrosis factor-
(TNF-
), are
immobilized to proteoglycans on the MC surface and MCP-1 and IL-8 are
secreted into the soluble phase, while the constitutive expression of
the CX3C chemokine fractalkine was not significantly
induced (38). Monocyte adhesion to activated MC involved
1- and
2-integrins, the GRO-
and IL-8
receptor CXCR2, and fractalkine. In contrast, the transendothelial
migration of monocytes toward MC was mediated by the MCP-1 receptor
CCR2 while CXCR2 played a minor role (38).
The nuclear factor-B (NF-
B)/Rel transcription factor family has
been shown to play a pivotal role for the activation and induction of
adhesion molecules and chemokines in MC by regulating expression at the
level of gene transcription (17). In resting cells,
inactive NF-
B forms a complex with its cytoplasmic inhibitor I
B-
, which retains the NF-
B complex in the cytoplasm by
masking its nuclear localization sequence. Upon stimulation, I
B-
is phosphorylated, ubiquitinated, and subsequently degraded via the proteosome pathway (11, 18, 24, 30). The proteosome is localized in the cytoplasm and nucleus, forming a 26S complex, and
contains a 20S proteolytic core responsible for the degradation and turnover of proteins (27). Unbound from its inhibitor,
the NF-
B heterodimer translocates to the nucleus, where it binds to
the promoter motifs of NF-
B-dependent genes that include ICAM-1, VCAM-1, MCP-1, and other proinflammatory molecules.
The inhibition of NF-B activation has been suggested to be of use in
suppressing the inflammatory response in vitro but also in animal
models of renal disease (17). Given the central role of MC
in promoting leukocyte infiltration during glomerular disease, we were
prompted to investigate the potential function of NF-
B as a central
mediator of glomerular leukocyte recruitment. In this study, we were
able to demonstrate that inhibition of I
B-
degradation by the
protease inhibitors N-
-tosyl-L-lysine
chloromethyl ketone (TLCK) and MG-132 or adenovirus-mediated I
B-
overexpression in MC impaired the upregulation of adhesion molecule and
chemokine expression in MC in response to TNF-
. This was associated
with decreased firm adhesion of monocytic cells to activated MC and transendothelial migration of monocytes toward stimulated MC. Thus
these data demonstrate that modulation of NF-
B activity may be a
potential target in the control of glomerular leukocyte infiltration.
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MATERIALS AND METHODS |
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Cell culture and reagents. Primary human MC (Clonetics) were used at passages 3-9 and cultured in basal MC medium (Clonetics), supplemented with 5% FCS and 50 mg/ml of gentamicin (GIBCO BRL). Mono Mac 6 cells (from Dr H. W. Ziegler-Heitbrock) were maintained as described (33, 40). Human umbilical vein endothelial cells (HUVEC) were used at passages 2-4 and grown in low serum PromoCell medium (33). Reagents were from Sigma Chemical unless otherwise stated.
Overexpression of adenoviral-encoded IB-
.
Construction of the adenoviral vector encoding for I
B-
(rAd.I
B-
; a kind gift from Dr. R. de Martin, Vienna International Research Center), and infection was performed as previously described (37). Briefly, subconfluent MC were washed with
phosphate-buffered saline (PBS) and incubated with the adenovirus in
PBS for 30 min at 37°C, washed, and then cultured for 48 h. An
equivalent infection with a control adenoviral vector (8)
encoding green fluorescence protein (rAd.GFP) was performed at
different multiplicities of infection (MOI). GFP expression was
analyzed by flow cytometry as described (8), and the most
effective transduction rate was achieved at a MOI of 300. Overexpression of I
B-
may induce apoptosis in
TNF-
-activated MC by suppressing induction of inhibitor of
apoptosis proteins (29); however, cell viability
was determined to be >95% under all conditions.
Western blot analysis.
Cells were left untreated, pretreated for 2 h with TLCK (50 µM)
or MG-132 (20 µM) or transfected with IB-
, left in culture for
48 h, and then activated with TNF-
(100 U/ml, 20 min).
Cells were lysed in sample buffer containing protease inhibitors, and lysates were separated by 12.5% SDS-PAGE. Proteins were transferred to
nitrocellulose membranes and reacted with a monoclonal antibody (MAb)
to I
B-
(Santa Cruz Biotechnology). Blots were developed using
chemiluminescence (ECL, Amersham).
Flow cytometry. Confluent MC were trypsinized, washed, and reacted with saturating concentrations of MAb for 30 min on ice, washed and stained with fluorescein isothiocyanate (FITC)-conjugated IgG (Boehringer Mannheim), and washed and analyzed in a FACScan (Becton Dickinson) (34, 35).
RT-PCR.
Total RNA was isolated by phenol/chloroform/isoamylalcohol extraction,
and cDNA was reverse transcribed from 2 µg of RNA. PCR was
performed, and PCR products were analyzed by agarose gel electrophoresis and quantitated by HPLC as described (33,
34). Primer sequences were as published for ICAM-1, VCAM-1,
-actin (34), GRO-
, MCP-1 (35), and
fractalkine (4), and were TACTCCAAACCTTTCCACCC (IL-8
sense) and AACTTCTCCACAACCCTCTG (IL-8 antisense).
Quantification of protein.
The concentration of MCP-1 and GRO- protein present in the MC
supernatants was determined using a sandwich ELISA (R&D Systems) performed according to the manufacturer's protocols. IL-8
concentrations were measured using antibodies from R&D Systems,
following their protocol for a double-ligand ELISA.
Immunofluorescence. MC were grown to confluence on glass coverslips. Cells were fixed in 3.7% formaldehyde, and coverslips were incubated for 2 h at RT with 10% heat-inactivated human serum in PBS to block nonspecific binding. The cells were incubated with the primary antibody for 30 min at RT, washed, and then incubated with FITC-conjugated IgG for 30 min at RT. Coverslips were allowed to air dry and were then mounted with Mowiol. During inspection and analysis using a Leica DMRBE fluorescence microscope with a ×100 oil-immersion objective, images were recorded on Kodak 400ASA film.
Monocyte adhesion on MC in shear flow. Laminar flow assays were performed as previously described (16, 23, 32). MC were grown to confluence in 35-mm petri dishes that were assembled as the lower wall in a parallel wall flow chamber and mounted on the stage of an Olympus IMT-2 inverted microscope with ×20 and ×40 phase-contrast objectives. Mono Mac 6 cells (106/ml) suspended in Hanks' balanced salt solution containing 10 mmol/l HEPES, pH 7.4, 0.5% human serum albumin, 1 mmol/l Mg2+, and 1 mmol/l Ca2+ (added shortly before the assay) were kept in a heating block at 37°C during assays and perfused into the flow chamber at a rate of 1.5 dyn/cm2 for 5 min. The number of firmly adherent cells after 5 min was quantitated in multiple fields (at least 5/experiment) by analysis of images, recorded with a long integration JVC 3CCD video camera and a JVC SR L 900 E video recorder, and expressed as cells/mm2. The type of adhesion analyzed was restricted to primary, i.e., direct interactions of monocytes with endothelium. Consistent with findings using a similar flow chamber (16), secondary attachment to already adherent monocytes, which is mediated by L-selectin and results in the formation of linear strings (1), occurred only sporadically at 1.5 dyn/cm2, accounting for a very small component of total interactions. Data are expressed as means ± SD, and statistical significance was determined by analysis of variance.
Chemotaxis assays. HUVEC were grown on 6.5-mm Transwell inserts (Costar, 8-µm pore size). Otherwise, inserts were untreated. MC were grown to confluence in the bottom chamber of the 24-well plate. Transwells were inserted, and monocytes in assay medium (RPMI 1640, medium 199, 0.5% human serum albumin) were added to the top chamber. A dilution of cells served as a measure of input. Cells were allowed to transmigrate for 2 h. Input and migrated cells were counted by flow cytometry with light scatter gates for monocytes.
Statistics. Statistical significance was determined by analysis of variance, and differences with P < 0.05 were considered to be significant.
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RESULTS |
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TLCK, MG-132, or
IB-
overexpression inhibits
TNF-
-induced degradation of I
B-
in MC.
Activation and nuclear translocation of NF-
B requires
phosphorylation, ubiquitination, and subsequent degradation of its cytoplasmic inhibitor I
B-
via the I
B kinase and the proteosome pathway (11, 18, 24, 30). To confirm the effects of
protease inhibitors on TNF-
-induced I
B-
degradation in MC,
I
B-
protein was detected by immunoblotting. Unstimulated MC
displayed a basal level of I
B-
, which is almost completely
degraded after TNF-
stimulation for 20 min (Fig.
1A). MC were
pretreated with TLCK (11) or the proteosome
inhibitor MG-132 (24) for 2 h and were then
challenged with TNF-
for 20 min in the continuing presence of the
inhibitors. This prevented TNF-
-induced I
B-
degradation, although TLCK appeared to be less effective (Fig. 1A).
Adenoviral overexpression of I
B-
in HUVEC has been shown to
prevent complete I
B-
degradation induced by TNF-
(35). To investigate whether adenoviral gene transfer of
I
B-
exerts similar protective effects on I
B-
in MC, Western
blotting of transfected MC with adenoviral-encoded I
B-
was
performed. Overexpression of I
B-
in MC, but not infection with
the control adenoviral vector rAd.GFP, increased basal levels of
I
B-
(Fig. 1B) and prevented complete I
B-
degradation in response to TNF-
stimulation, as opposed to untreated
MC (Fig. 1A). These data suggest that TLCK and MG-132
treatment of MC and cytoplasmic I
B-
overexpression potently
inhibit the TNF-
-induced initiation of NF-
B activation in MC.
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Inhibition of NF-B reduces mRNA expression of inflammatory
proteins induced by TNF-
.
NF-
B translocated to the nucleus binds to promoter regions of
NF-
B-dependent genes, regulating the transcriptional activity of
many adhesion molecules and chemokines involved in monocyte adhesion
and transmigration (17). To assess the effects of
inhibited I
B-
degradation on transcriptional activity, RT-PCR
with subsequent HPLC quantification was performed. As previously
described, TNF-
stimulation of MC for 4 h markedly upregulated
mRNA levels of the adhesion molecules ICAM-1 and VCAM-1 and the
chemokines MCP-1, IL-8, and GRO-
, but only slightly increased mRNA
levels of fractalkine (Fig. 2).
Pretreatment of MC with TLCK or MG-132 or rAd.I
B-
overexpression,
but not infection with the control adenoviral vector rAd.GFP, inhibited
this upregulation in mRNA transcripts (Fig. 2). MG-132, which exerted
stronger inhibitory effects on I
B-
degradation than TLCK or
I
B-
overexpression, also more effectively blocked transcriptional
activation of adhesion molecules and chemokines (Fig. 2). Thus
inhibition of I
B-
degradation is paralleled by impaired
transcriptional upregulation of adhesion molecules and chemokines in
response to TNF-
stimulation.
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TNF--induced surface expression of
MC adhesion molecules is inhibited by
TLCK, MG-132, or overexpression of
I
B-
.
To investigate whether suppression of transcriptional activity was
associated with decreased surface protein expression of the
immunoglobulin family members ICAM-1 and VCAM-1, flow cytometry was
performed. As previously shown, MC express basal levels of ICAM-1 and
low levels of VCAM-1 (Fig. 3), and
adhesion molecule expression was markedly increased after TNF-
treatment for 4 h (Fig. 3). In parallel with mRNA expression of
transcripts, pretreatment of MC with TLCK or MG-132 almost completely
inhibited the TNF-
-induced upregulation of ICAM-1 and VCAM-1 (Fig.
3, A-D). Similarly, rAd.I
B-
transfection of MC
inhibited the TNF-
-induced increase of ICAM-1 and VCAM-1 surface
expression (Fig. 3, E and F).
|
TNF--induced expression of
GRO-
, MCP-1, and IL-8
is inhibited by TLCK, MG-132, or
overexpression of I
B-
.
We have previously shown that TNF-
stimulation of MC induces the
immobilization of GRO-
and IL-8 on the MC surface via
heparan proteoglycan binding, whereas MCP-1 but also IL-8 are
secreted as soluble proteins (38). We investigated effects
of TLCK, MG-132, and I
B-
overexpression on the TNF-
-induced
chemokine expression. Immunofluorescence staining of MC with specific
antibodies was used to detect surface-associated proteins. As
previously described, TNF-
stimulation of MC for 4 h induced
the surface association of GRO-
and IL-8 (Fig.
4A, not shown), which was
inhibited by preincubation of MC with TLCK, MG-132, or I
B-
overexpression (Fig. 4A, data not shown). Chemokines
secreted as soluble proteins into the supernatants were measured by
ELISA. Compared with untreated MC, supernatants of TNF-
-stimulated
MC revealed increased amounts of soluble GRO-
, and foremost MCP-1
and IL-8. Pretreatment of MC with TLCK and mostly MG-132 or
transduction of MC with the I
B-
adenovirus inhibited the
secretion of chemokines induced by TNF-
(Fig. 4B, not
shown). These results indicate that by preventing I
B-
degradation
and NF-
B-dependent transcription of adhesion molecules and
chemokines, TLCK, MG-132, and cytoplasmic I
B-
overexpression
prevented the expression of adhesion molecules and inhibited chemokine
secretion and immobilization on the MC surface in response to TNF-
.
|
Transmigration toward stimulated MC.
To further investigate the effects of TLCK, MG-132, or IB-
transfection on transmigration of monocytes, two-chamber transmigration assays were performed. Monocytes added to the top chamber were allowed
to transmigrate across HUVEC grown on inserts toward untreated or
TNF-
-stimulated MC for 2 h. TNF-
stimulation elicited a
twofold increase in the number of transmigrated cells compared with
untreated cells, and pretreatment of MC with TLCK or MG-132 or
I
B-
overexpression of MC substantially inhibited the
TNF-
-induced transmigration (Fig.
5A, not shown).
|
Monocyte adhesion to TNF--stimulated
MC.
Glomerular monocyte infiltration occurs during the pathogenesis of
numerous kidney diseases, and MC have been critically involved through
expression of adhesion molecules and chemokines (7, 12, 20, 21,
26, 27). We have recently shown that monocyte adhesion to
activated MC is mediated by the
2- and
4-integrins interacting with ICAM-1 and VCAM-1 on MC,
that the GRO-
receptor CXCR2 contributes to firm monocyte arrest on
stimulated MC, and that a gradient of soluble MCP-1 predominantly
mediates transmigration of monocytes toward MC (38). To
study whether pretreatment of MC with TLCK, MG-132, or I
B-
overexpression also impairs the TNF-
-induced adhesion of monocytic
cells to stimulated MC, as a result of reduced expression of adhesion
molecules and chemokines, monocyte adhesion was studied under flow
conditions as a measure of firm arrest. Monocytic Mono Mac 6 cells,
which express a similar array of adhesion molecules and chemokine
receptors as monocytes (40), were perfused over MC at a
constant shear rate of 1.5 dyn/cm2, and after a period of
accumulation, the number of firmly attached cells was determined.
Compared with untreated MC, TNF-
treatment markedly increased the
number of firmly adherent Mono Mac 6 cells. Preincubation of MC with
TLCK or MG-132, as well as I
B-
overexpression but not infection
with the control adenoviral vector rAd.GFP before TNF-
stimulation,
significantly inhibited the number of cells firmly attached and reduced
the monocyte adhesion to almost background level (Fig. 5B).
These data show that inhibited adhesion molecule and chemokine
expression after TLCK, MG-132, and I
B-
overexpression is
associated with a decrease in firm monocyte adhesion to stimulated MC
and also inhibited the TNF-
-induced transmigration of monocytes toward stimulated MC.
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DISCUSSION |
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NF-B is a family of dimeric proteins that belongs to the Rel
family. The Rel homology domain determines dimerization, nuclear localization, and binding to the
B element in the promoter and enhancer regions of many target genes (18). These include
genes encoding for adhesion molecules and chemokines and cytokines that are involved in the inflammatory and proliferative responses of MC
(17). NF-
B activation has been investigated and
confirmed to occur in vitro through well-known inducers of NF-
B such
as TNF-
, IL-1
, and immunoglobulin proteins and in vivo in several animal models of renal disease such as antiglomerular basement membrane
or immune complex nephritis (17). Moreover, inhibitors of
NF-
B activation such as pyrrolidine dithiocarbamate,
corticoids, antioxidants, or angiotensin-converting enzyme
inhibitors have been shown to prevent the activation of
NF-
B and subsequent mRNA expression of several adhesion molecules
and chemokines (2, 15, 17, 25, 27).
In this study, we investigated possible effects of TLCK, MG-132, and
IB-
overexpression on the NF-
B-dependent adhesion molecules
ICAM-1 and VCAM-1 and the chemokines GRO-
, MCP-1, and IL-8, and
subsequently on monocyte recruitment to MC. TLCK, an irreversible
inhibitor of serine proteases, has been shown to inhibit
I
B-
degradation and NF-
B activation in endothelial cells
(11). MG-132 has been described to inhibit the proteolytic activity of the 20S and 26S proteosomal complex, thus blocking proteosomal I
B-
degradation and reducing the accumulation of NF-
B in the nucleus (24). Associated with decreased
expression of adhesion molecules and chemokines in endothelial cells,
the activation of NF-
B has been suggested to be a critical target for therapeutic intervention (24). Ectopic expression of
I
B-
in endothelial cells, achieved by recombinant adenoviral
transfection, has been shown to prevent NF-
B activation, and,
correspondingly reduced expression of adhesion molecules and
chemokines, thus inhibiting monocyte recruitment to activated
endothelium (35). Although adenoviral gene transfer itself
may result in an inflammatory response and antigenicity
(15), localized applications have been demonstrated to be
feasible in the kidney (5, 39) and glomerular cells
(10) and may be of potential use in limiting the
inflammatory response in designated areas.
We have shown that in MC inhibition of IB-
degradation by the
inhibitor of trypsin TLCK, the proteosome inhibitor MG-132 and
overexpression of the cytoplasmic NF-
B inhibitor I
B-
can reduce the TNF-
-induced transcriptional upregulation and surface expression or secretion of adhesion molecules and chemokines. In
functionally relevant systems, this was associated with a decrease in
adhesion and transmigration of monocytes on stimulated MC.
To interfere with early signaling events in response to TNF-, cells
must be exposed to MG-132 either before or simultaneously with TNF-
,
as has been shown for E-selectin expression on endothelial cells
(24). Pretreatment of MC for 2 h before TNF-
challenge almost completely inhibited the TNF-
-induced degradation
of I
B-
and abolished the TNF-
-induced increase in expression
of the adhesion molecules ICAM-1 and VCAM-1 and the chemokines GRO-
, MCP-1, and IL-8. TLCK pretreatment of MC compared with MG-132 exerted
weaker inhibitory effects, but still effectively prevented the
TNF-
-elicited I
B-
degradation and reduced adhesion molecule and chemokine expression. In MC transfection with adenoviral-encoded I
B-
, TNF-
stimulation decreased the levels of I
B-
slightly, but it remained markedly higher than in TNF-
-treated cells
and also reduced adhesion molecule and chemokine expression. I
B-
expressed by adenoviral gene transfer may be phosphorylated and subsequently degraded, but as observed with lipopolysaccharide stimulation (37), the overwhelming proportion of I
B-
could not be processed. This may, in part, be due to the presence of the nuclear localization sequence, but may also reflect a high efficiency of infection by the adenovirus.
Leukocyte adhesion was shown to involve the 2- and
4-integrins interacting with ICAM-1 and VCAM-1,
respectively (16, 28). Moreover, we have demonstrated that
a combination of MAb blocking
4- and
2-integrins inhibited monocyte adhesion on
stimulated MC in shear flow (38). Thus effects of TLCK,
MG-132, and overexpression of I
B-
on monocyte adhesion are likely
to be at least in part due to the reduced expression of ICAM-1 and
VCAM-1 on MC. Inhibition of NF-
B suppressed the production of the
chemokines GRO-
, MCP-1, and IL-8 in TNF-
-stimulated MC. GRO-
and IL-8 can be immobilized on the surface of activated MC by heparan
proteoglycans, and through this, have been demonstrated to be
critically involved in firm monocyte adhesion on activated MC, while
MCP-1 but also IL-8 were secreted as soluble molecules, thus
establishing and maintaining a chemokine gradient required for
triggering subsequent transmigration (38). We were able to
show that the TNF-
-induced immobilization of GRO-
and IL-8 on the
MC surface was almost abolished by preincubation of MC with TLCK,
MG-132, or I
B-
overexpression, and, similarly, secretion of MCP-1
and IL-8 from activated MC was markedly inhibited by TLCK, MG-132, and
I
B-
transfection. In addition to the reduction of adhesion
molecule expression, the marked decrease in GRO-
and IL-8 surface
association could be responsible for the inhibition of monocyte arrest
seen in MC preincubated with TLCK, MG-132, and transfected with
I
B-
. Because the impaired secretion of chemokines could be
responsible for reduced transmigration, our data provide a mechanistic
clue of the pluripotent effects of this concept on the machinery for
glomerular monocyte recruitment.
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ACKNOWLEDGEMENTS |
---|
The authors thank Prof. P. C. Weber for continuous support, T. Rubic, B. Zimmer, and P. Weinert for expert help, and R. de Martin for adenoviral work. This work fulfills, in part, the requirements for the doctoral thesis of A. Zernecke.
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FOOTNOTES |
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This work was supported by Deutsche Forschungsgemeinschaft Grants WE1913/1-2 and GRK 438.
Address for reprint requests and other correspondence: C. Weber, Dept. of Molecular Cardiovascular Research, Univ. Hospital Aachen, Pauwelsstrasse 30, D-52074 Aachen, Germany.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 March 2001; accepted in final form 1 August 2001.
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