Am J Physiol Renal Physiol 293: F1262–F1271, 2007.
First published August 1, 2007; doi:10.1152/ajprenal.00445.2006.
The role of ICAM-1 in endotoxin-induced acute renal failure
Xiaoyan Wu, Rongqing Guo, Ying Wang, and Patrick N. Cunningham
Section of Nephrology, University of Chicago, Chicago, Illinois
Submitted 9 November 2006; accepted in final form 27 July 2007
sepsis; neutrophils; vascular cell adhesion molecule-1; chemokines
within sites of inflammation (1, 20, 29, 34). In previous work,
we confirmed that administration of LPS causes an influx of
neutrophils into the kidney in parallel with a decline in renal
function. Interventions that protected against LPS-induced
ARF, such as interruption of TNFR1 signaling or use of
caspase inhibitors, also led to a decrease in neutrophil infiltration in parallel with a decrease in renal expression of MIP-2
and ICAM-1 (7, 9, 15). These findings suggest that this influx
of neutrophils may contribute to the renal functional and
structural injury that is seen in response to LPS.
In particular, the adhesion molecule ICAM-1 has been found
to be important to the firm adhesion of neutrophils and other
leukocytes to the endothelium, allowing their attachment before their potential traffic between adjacent endothelial cells to
reach underlying structures. ICAM-1 is expressed mainly on
endothelial cell surfaces, interacting with two principal ligands,
␤2 integrins LFA-1 (CD11a/CD18) and Mac-1 (CD11b/
CD18), present on circulating leukocytes (12, 24). ICAM-1 has
been shown by others to be acutely upregulated in kidney after
LPS administration, primarily on the renal endothelium, but
also in renal tubules (28). Mice lacking expression of ICAM-1
have reduced mortality in response to septic shock (59, 64).
Furthermore, mice deficient in ICAM-1 experienced less renal
leukocyte infiltration and reduced functional and structural
injury in response to ischemia-reperfusion injury (IRI) (23),
with similar protection observed with the use of ICAM-1 or
CD11/CD18 neutralizing Abs (22). Taken together, this background suggests that ICAM-1-mediated neutrophilic infiltration may have a pathogenic role in LPS-induced ARF. Therefore, the following study was performed to address this hypothesis.
ACUTE RENAL FAILURE (ARF) is a common result of sepsis and
septic shock and is responsible for significant morbidity and
mortality (52). Despite its prevalence, therapeutic options for
treating this condition are limited to supportive interventions
such as dialysis. One animal model that has offered insight into
the pathogenesis of septic ARF is that of LPS-induced ARF
(53). While many bacterial products may contribute to renal
injury, LPS, a component of the outer cell membrane of
gram-negative bacteria, reproduces most of the clinical sequellae of sepsis when injected into animals, including ARF.
LPS-induced ARF is associated with mild and patchy areas of
tubular injury similar to what is seen in human ARF (8, 56).
LPS administration is well-known to trigger an influx of
neutrophils into various organs, such as lung and liver (1, 20).
This process is directed by the systemic release of chemotactic
chemokines such as macrophage inflammatory protein-2
(MIP-2) and keratinocyte-derived chemokine (KC) and the
local expression of adhesion molecules such as E- and Pselectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) on endothelium
ARF model. Three strains of male mice were studied: mice lacking
expression of ICAM-1 (ICAM-1⫺/⫺), mice deficient in CD18 (CD18def), and wild-type (C57BL/6J) mice. CD18-def mice are a hypomorphic strain that express CD11a/CD18 and CD11b/CD18 at a level that
is ⬃2% of normal, increasing to 16% of normal after activation of
neutrophils with PMA (62). ICAM-1⫺/⫺ and CD18-def mice had both
been backcrossed for ⬎10 generations on a C57BL/6J background.
All mice were obtained commercially and studied at 9 wk of age
(Jackson Laboratories, Bar Harbor, ME), n ⫽ 8 per group. Mice
received a single intraperitoneal injection of 6 mg/kg Escherichia coli
LPS (Sigma, St. Louis, MO) at time 0. Blood was obtained for blood
urea nitrogen (BUN) and cytokine measurements at time of LPS
injection, and 2, 24, and 48 h later under continuous isoflurane/oxygen
anesthesia. Mice were killed 48 h after LPS injection, with harvest of
blood and kidney tissue. As controls, ICAM-1⫺/⫺, CD18-def, and
wild-type mice were sham-injected with 150 mM saline and their
blood and tissue were harvested as above (n ⫽ 5 per group). For time
Address for reprint requests and other correspondence: P. N. Cunningham,
MC 5100, Section of Nephrology, Univ. of Chicago, 5841 South Maryland
Ave., Chicago, IL 60637 (e-mail: [email protected]).
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.
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METHODS
0363-6127/07 $8.00 Copyright © 2007 the American Physiological Society
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Wu X, Guo R, Wang Y, Cunningham PN. The role of ICAM-1
in endotoxin-induced acute renal failure. Am J Physiol Renal Physiol 293: F1262–F1271, 2007. First published August 1, 2007;
doi:10.1152/ajprenal.00445.2006.—The pathogenesis of acute renal
failure (ARF) occurring during the course of sepsis is incompletely
understood. Intercellular adhesion molecule-1 (ICAM-1) is a key cell
adhesion molecule upregulated by LPS, which binds to the integrins
CD11a/CD18 and CD11b/CD18 present on the surface of leukocytes.
We hypothesized that ICAM-1 facilitates renal injury in LPS-induced
ARF. To test this, three groups of mice (n ⫽ 8 per group) were
injected intraperitoneally with 6 mg/kg LPS: 1) normal C57BL/6
mice, 2) mice with a targeted deficiency of ICAM-1 (ICAM-1⫺/⫺),
and 3) mice expressing very low levels of CD18 (CD18-def). ICAM1⫺/⫺ mice were significantly resistant to LPS-mediated ARF, as
opposed to CD18-def mice, which developed severe ARF, as did
wild-type controls (48 h blood urea nitrogen 143 ⫾ 31.5, 70.8 ⫾ 24.4,
and 185 ⫾ 16.6 mg/dl in wild-type, ICAM-1⫺/⫺, and CD18-def mice,
respectively, P ⬍ 0.05). At death, ICAM-1⫺/⫺ mice had significantly
less renal neutrophil infiltration than the other two groups, as well as
less histological tubular injury. Depletion of neutrophils with mAb
Gr-1 led to a profound exaggeration of tumor necrosis factor (TNF)
release and high mortality, but neutrophil-depleted mice receiving
10-fold less LPS were protected against ARF despite TNF release
similar to what is normally associated with LPS-induced ARF. LPS
caused a significant increase in renal expression of chemokines;
however, this increase was significantly exaggerated in CD18-def
mice, which may account for their lack of protection. In conclusion,
these data show that ICAM-1 plays a key role in LPS-induced ARF.
ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
AJP-Renal Physiol • VOL
Cytokine measurement. TNF levels were determined from sera
obtained at 0 and 2 h after LPS administration using commercially
available ELISA kits for mouse TNF (R&D Systems, Minneapolis,
MN), according to the manufacturer’s instructions, on serum
diluted 1:10.
Real-time PCR. A portion of frozen kidney was placed in TRIzol
reagent (GIBCO BRL, Grand Island, NY), from which total RNA was
purified according to the manufacturer’s instructions. To remove all
traces of genomic DNA, samples were then treated with RNAse-free
RQ1 DNAse (Promega, Madison, WI; 1 U per 4 ␮g RNA) in 10-␮l
reaction buffer (final concentration 40 mM Tris 䡠 HCl, 10 mM MgSO4,
1 mM CaCl2, pH 8.0) at 37°C for 30 min. This was followed by
addition of 1 ␮l of 20 mM EGTA, pH 8.0, to stop the reaction, and
incubation at 65°C for 10 min to inactivate the DNAse. cDNA was
generated from RNA using random hexamers as primers with the
SuperScript first-strand synthesis kit (GIBCO BRL), according to the
manufacturer’s instructions, and diluted fivefold before analysis.
Real-time PCR was performed using the Prism 7700 reactor and
the SybrGreen intercalating dye method with HotStar DNA polymerase (Applied Biosystems, Foster City, CA). Each reaction was conducted in a total volume of 50 ␮l with primers at 200 nM, 1 mM
dNTPs, 3 mM MgCl2, and 10 ␮l of sample or standard cDNA. PCR
was carried out with a hot start at 95°C (5 min) followed by 45 cycles
at 95°C (15 s)/60°C (30 s). For each sample, the number of cycles
required to generate a given threshold signal (Ct) was recorded. Using
a standard curve generated from serial dilutions of kidney cDNA, the
ratio of ICAM-1 expression relative to GAPDH expression was
calculated for each experimental animal and normalized relative to an
average of ratios from the control (no LPS) group. Measurements of
KC, MIP-2, and VCAM-1 mRNA expression were performed in an
analogous fashion. Products of each reaction yielded a single band
when run on agarose gel, confirming specific amplification. Primers
were synthesized by Integrated DNA Technologies (Coralville, IA),
with sequences as follows: GAPDH forward primer 5⬘-GGC AAA
TTC AAC GGC ACA GT-3⬘, GAPDH reverse primer 5⬘-AGA TGG
TGA TGG GCT TCC C-3⬘, MIP-2 forward primer 5⬘-CAC CAA
CCA CCA GGC TAC A-3⬘, MIP-2 reverse primer 5⬘-GCC CTT GAG
AGT GGC TAT GA-3⬘, KC forward primer 5⬘-ACC CGC TCG CTT
CTC TGT-3⬘, KC reverse primer 5⬘-TGG CTA TGA CTT CGG TTT
GG-3⬘, VCAM-1 forward primer 5⬘-CCC GAA CTC CTT GCA CTC
TA-3⬘, and VCAM-1 reverse primer 5⬘-TGT GCC TCC ACC AGA
CTG TA-3⬘.
Statistics. Data were analyzed with Minitab software (State College, PA). Unless noted otherwise, data are given as means ⫾ SE.
Groups were compared by two-tailed t-test, or ANOVA when more
than two groups were compared. Tukey’s correction for multiple
comparisons was used when comparing more than two groups. Because expression data obtained by real-time PCR were not normally
distributed for any group, ANOVA was performed on log-transformed
data. A P value ⱕ0.05 was considered significant.
RESULTS
Induction of ICAM-1 within kidney. To gain insight into
changes in renal ICAM-1 expression soon after LPS is injected, when ARF is first established, wild-type mice were
killed at various time points after LPS injection, and their
kidneys were analyzed for ICAM-1 expression and neutrophil
infiltration. At baseline, ICAM-1 expression is low, limited to
weak staining in small arteries (Fig. 1, A-B). Likewise, few
neutrophils are seen in kidney at baseline. However, within 3 h
after LPS injection (6 mg/kg), both ICAM-1 mRNA expression, as quantitated by real-time PCR, and renal neutrophil
infiltration were significantly increased. This parallel increase
in renal ICAM-1 expression and neutrophil infiltration continued at 6 h and persisted at 48 h. Immunohistochemistry
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course experiments, wild-type mice were injected with LPS, and
kidney was harvested 0, 3, 6, and 12 h later for analysis of mRNA and
immunohistochemistry (n ⫽ 6 per group). BUN concentrations were
determined with a Beckman CX5CE autoanalyzer. All experiments
were done in accord with a written protocol approved by an independent review process conducted by the University of Chicago Institutional Animal Care and Use Committee.
Depletion of neutrophils and blockade of ICAM-1 and VCAM-1.
For neutrophil depletion experiments, rat mAb Gr-1 was purchased
(BD Pharmingen, San Diego, CA). Either Gr-1 or isotype control rat
IgG2b was injected at a dose of 0.2 mg/mouse ip into wild-type mice,
n ⫽ 10 per group. Blood was obtained at time of Gr-1 injection and
24 h later for circulating neutrophil counts, as determined by Coulter
counter, to confirm depletion. LPS was injected 24 h after Gr-1, and
blood was obtained at that time and 2, 6, and 24 h later, with death of
mice and harvest of tissue at 24 h after LPS. In an attempt to correct
for the exaggeration of TNF release seen with neutrophil depletion, a
third group of Gr-1-injected mice received low-dose LPS, 0.6 mg/kg,
n ⫽ 8, with a fourth comparison group receiving IgG followed by 0.6
mg/kg LPS.
In separate experiments to test the role of ICAM-1 and VCAM-1,
rat mAbs YN1/1.7.4 (18, 30) or M/K-2.7 (19, 38) were injected into
wild-type and CD18-def mice. Polyclonal rat IgG (Invitrogen, Carlsbad, CA) was injected into wild-type mice as a control group. Abs
were injected 2 h before LPS, at a dose of 10 mg/kg ip, n ⫽ 6 per
group. Blood was obtained at time of LPS injection and 2, 6, and 24 h
later, with death of mice and harvest of tissue at 24 h after LPS.
YN1/1.7.4 or M/K-2.7 hybridomas were obtained from American
Type Culture Collection (Manassas, VA) and grown in DMEM/10%
FBS with 0.05 mM ␤-mercaptoethanol. Supernatant reactivity against
vascular structures in frozen sections from LPS-injected mouse kidney was confirmed by immunohistochemistry, and IgG was purified
using protein G affinity chromatography and concentrated to ⬃8
mg/ml by Centriprep (Millipore, Billerica, MA). All injected Abs
were either purchased in 5 mg/ml no azide/low endotoxin form or
dialyzed into sterile 150 mM NaCl in endotoxin-free glassware.
Pathology. For routine histological analysis, kidneys were sectioned coronally, fixed in phosphate-buffered formalin, embedded in
paraffin, and stained with periodic acid-Schiff base (PAS). Histological sections for each animal were assigned a semiquantitative score
for tubular injury by a blinded observer, as previously described (7).
A blinded observer assigned a score ranging from 0 (no injury) to 3
(widespread injury) for each of 3 variables: tubular dilatation/flattening, tubular casts, and tubular degeneration/vacuolization. For each
animal, 10 cortical high-power fields (HPF) were examined at random. For each variable within each field, a score of 0 was assigned
when less than 5% of the tubules were affected, a score of 1 when
5–33% were affected, a score of 2 when 34 – 66% were affected, and
a score of 3 when over 66% were affected. For immunohistochemistry, tissue was immediately frozen in OCT compound at ⫺20°C.
Four-micrometer kidney cryostat sections were fixed with ether/
ethanol, incubated with 0.3% H2O2 for 30 min, and blocked with
dilute horse serum. Sections were stained for neutrophils by sequential
incubation with rat anti-mouse neutrophil (mAb 7/4, Serotec, Raleigh,
NC) at 1:60 for 30 min followed by horseradish peroxidase (HRP)conjugated rabbit anti-rat IgG (Sigma) at 1:60 for 30 min and
diaminobenzidine reagent (Vector Laboratories, Burlingame, CA) for
10 min. A blinded observer counted the number of neutrophils per
HPF and recorded the average of 10 fields for each sample. In an
analogous fashion, frozen sections were stained with rat-anti-mouse
VCAM-1 at 1:20, or hamster-anti-mouse ICAM-1 at 1:60, followed
by HRP-anti-rat IgG or biotinylated rabbit-anti-hamster IgG at 1:100
and streptavidin-HRP (Abs purchased from BD Pharmingen). Methyl
green counterstain was used as a final step with the ICAM-1 staining.
Sections were scored as to the extent and intensity of VCAM-1
staining on a semiquantitative score ranging from 0 (minimal inensity)
to 4 (maximum).
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ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
confirmed that at 48 h after LPS administration renal ICAM-1
was strongly increased in peritubular capillaries, arterioles, and
to a lesser extent within renal tubules (Fig. 1C).
Effects of neutrophil depletion in LPS-induced ARF. To
study the role of renal neutrophilic infiltration in LPS-induced
ARF, mice were injected with mAb Gr-1 to deplete circulating
neutrophils. Twenty-four hours after Gr-1 injection, circulating
neutrophil counts fell markedly, confirming depletion (84 ⫾ 25
vs. 570 ⫾ 170 PMN/␮l in Gr-1 vs. control IgG group, P ⬍
0.05). At this time point, LPS was injected at a dose of 6
mg/kg. Despite near absence of circulating neutrophils, Gr-1treated mice experienced markedly increased mortality within
24 h (70 vs. 0%) and no protection against the ensuing ARF,
with significantly higher BUN at 6 h after LPS (48.0 ⫾ 7.9 vs.
39.5 ⫾ 1.3 mg/dl, P ⬍ 0.05; Fig. 2A). As expected, Gr-1injected mice had significantly fewer neutrophils present in
kidney (1.4 ⫾ 0.8 vs. 11.4 ⫾ 1.3 PMN/HPF, P ⬍ 0.01; Fig.
2B). However, neutrophil-depleted mice had a profoundly
exaggerated increase in serum TNF 2 h after LPS (16.2 ⫾ 2.2
vs. 2.7 ⫾ 0.6 ng/ml, P ⬍ 0.01; Fig. 2C) compared with control
mice injected with IgG. Given the central role of TNF in
LPS-induced ARF that has been previously demonstrated (7),
and the fact that LPS exerts most of its renal effects indirectly,
AJP-Renal Physiol • VOL
through TNF (9), we attempted to reduce this confounding
effect of neutrophil depletion on TNF release by injecting
neutrophil-depleted mice with a 10-fold lower dose of LPS, 0.6
mg/kg. Although Gr-1 still exaggerated LPS-induced TNF
release compared with IgG controls (Fig. 2C), these neutrophildepleted, low-dose LPS mice had 2-h TNF levels similar to
non-neutrophil-depleted, high-dose LPS mice (2.2 ⫾ 0.2 ng/
ml; Fig. 2C). Despite this similar, relatively high level of TNF,
these mice underwent a markedly lesser elevation in BUN at
24 h (32.6 ⫾ 0.5 mg/dl; Fig. 2A), with minimal renal neutrophil
infiltration and lesser histological injury. This minimal level of
ARF and renal neutrophil infiltration in the Gr-1 ⫹ low-dose
LPS group was similar to what was seen in IgG ⫹ low-dose
LPS (data not shown), despite a significantly greater elevation
in TNF (2.2 ⫾ 0.2 vs. 0.48 ⫾ 0.9 ng/ml, P ⬍ 0.01; Fig. 2C).
Role of ICAM-1 in renal injury. To study the pathogenic role
of ICAM-1 in LPS-induced ARF, ICAM-1⫺/⫺ and wild-type
mice were studied. Because the principal ligands for ICAM-1
are CD11a/CD18 and CD11b/CD18, mice deficient in CD18
(CD18-def) were also studied in parallel. These mice are not
absolutely deficient in CD18, but their granulocytes express
CD18 at levels that are only 2% of normal, increasing to 16%
of normal after stimulation of neutrophils with PMA (62).
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Fig. 1. Time course of intercellular adhesion molecule-1 (ICAM-1) expression and neutrophilic infiltration following administration of LPS. A: in wild-type
(WT) mice, LPS caused an acute rise in renal ICAM-1 expression as detected via real-time PCR, increasing in parallel with renal neutrophil infiltration; n ⫽
6 at each time point. B: at baseline, expression of ICAM-1 is low in WT mice, consisting only of weak expression in small arteries, with no detectable staining
in tubules or peritubular capillaries. C: injection of LPS stimulates strong expression of ICAM-1 in peritubular capillaries at 48 h, in addition to small arteries
(inset) and some tubular expression. D: negative control, with primary Ab omitted, is shown for comparison. Magnification: ⫻200, methyl green counterstain.
ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
Reduced expression of CD18 leads to a parallel decrease in
CD11a and CD11b expression due to degradation, and these
mice are phenotypically associated with deficiencies in neutrophil chemotaxis in response to intraperitoneal injection of
thioglycollate (62). After injection with LPS (6 mg/kg), all
mice experienced an acute rise in BUN indicative of ARF.
While this rise was severe in wild-type mice, the level of BUN
AJP-Renal Physiol • VOL
elevation in ICAM-1⫺/⫺ mice was significantly less (143 ⫾
31.5 vs. 70.8 ⫾ 24.4 mg/dl at 48 h, in wild-type vs. ICAM1⫺/⫺ mice, P ⬍ 0.05), demonstrating relative protection
against LPS-induced ARF (Fig. 3). The levels of BUN in the
CD18-def group were actually greater than that seen in wildtype mice, although not to a statistically significant degree
(185 ⫾ 16.6 mg/dl at 48 h). In addition to this decline in renal
function, LPS induced modest renal tubular injury, most prominent in the renal cortex, as has been previously described (8).
ICAM-1⫺/⫺ mice were also significantly protected against this
pathologic injury (Fig. 4), as quantitated by cortical tubular
injury scores (2.8 ⫾ 0.6, 1.4 ⫾ 0.4, and 3.2 ⫾ 0.4 in wild-type,
ICAM-1⫺/⫺, and CD18-def mice, respectively, P ⬍ 0.05 vs.
CD18-def mice and P ⫽ 0.13 vs. wild-type mice). Thus
ICAM-1 deficiency conferred protection against both the functional and structural injury caused by LPS.
Neutrophilic infiltration. We hypothesized that mice deficient in ICAM-1 would have impaired leukocyte trafficking
within the kidney following LPS. At baseline, neutrophils were
sparse in the kidney and equivalent between wild-type, ICAM1⫺/⫺, and CD18-def mice. As previously described, LPS administration induced a robust infiltration of the kidney with
neutrophils (Fig. 5, A-B). In contrast to the concentration of
neutrophils observed at the corticomedullary border seen in
renal ischemia-reperfusion (22), neutrophils were distributed in
a patchy manner throughout the renal cortex, and to a lesser
extent within the renal medulla. In contrast to both wild-type
and CD18-def mice, ICAM-1⫺/⫺ mice had significantly fewer
neutrophils within the kidney 48 h after LPS infiltration
(10.2 ⫾ 1.0, 4.6 ⫾ 0.8, and 9.9 ⫾ 1.0 PMN/HPF in wild-type,
ICAM-1⫺/⫺, and CD18-def mice, respectively, P ⬍ 0.05
for ICAM-1⫺/⫺ vs. other groups; Fig. 5, C-D). Thus deficiency
of ICAM-1, but not CD18, protected against LPS-induced
neutrophilic infiltration.
Systemic and renal inflammation. We previously demonstrated the central role of TNF acting through its receptor
TNFR1 in LPS-induced ARF (7). Therefore, we wanted to
confirm that any protective role associated with ICAM-1 deficiency was not mediated through a difference in TNF activity.
As expected, LPS administration caused a profound increase in
circulating TNF levels, rising from undetectable at baseline in
all three strains to a peak at 2 h. However, this was not
significantly different among the different groups (2.70 ⫾ 0.65,
2.03 ⫾ 0.49, and 1.62 ⫾ 0.17 ng/ml in wild-type, ICAM-1⫺/⫺,
and CD18-def mice, respectively, P ⫽ NS; Fig. 6) and thus
Fig. 3. Renal function following administration of LPS. LPS caused an acute
rise in serum BUN in all groups, but this was significantly less in ICAM-1⫺/⫺
mice. *P ⬍ 0.05 vs. WT and mice deficient in CD18 (CD18-def) groups.
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Fig. 2. Renal dysfunction and tumor necrosis factor (TNF) release following
LPS administration to neutrophil-depleted mice. A: neutrophil-depleted mice
were not protected against LPS-induced acute renal failure (ARF), with
significantly higher blood urea nitrogen (BUN) 6 h after LPS injection; n ⫽ 10
per group. B: mice depleted of neutrophils with mAb Gr-1 had minimal
neutrophil infiltration into kidney following LPS, in contrast to the robust
infiltration of kidney seen after LPS in the control (non-neutrophil-depleted)
group. C: serum TNF, which was undetectable at baseline, increased strongly
in all mice 2 h after LPS injection. However, neutrophil-depleted mice had a
profound exaggeration in this TNF release, associated with high mortality.
Injection with 10-fold lower dose of LPS (0.6 mg/kg) in Gr-1-treated mice led
to serum TNF levels similar to what was seen with high-dose LPS in
non-neutrophil-depleted mice; n ⫽ 8. However, neutrophil depletion in mice
receiving low-dose LPS led to protection against neutrophil influx and BUN
elevation, despite similar levels of TNF release (A-B). *P ⬍ 0.05, **P ⬍ 0.01
vs. IgG ⫹ 6 mg/kg LPS control group. ***P ⬍ 0.01 vs. IgG ⫹ 0.6 mg/kg LPS
control group.
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ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
cannot explain to the resistance to LPS-induced ARF seen in
the ICAM-1⫺/⫺ group.
Given that CD11a/CD18 and CD11b/CD18 are the only
known ligands through which leukocytes bind to ICAM-1, it
was notable that CD18-def mice were not protected against the
neutrophil infiltration or ARF induced by LPS. Other investigators working with adhesion molecules have also noted inconsistencies with results obtained with knockout strategies
and have implicated compensatory increases in other pathways
to account for these observations (13, 48, 61). To determine
whether a similar compensatory increase was present in the
CD18-def group, we assayed kidney tissue for expression of
neutrophil-attractant chemokines and VCAM-1 by real-time
PCR. As shown in Table 1, LPS caused a large increase in
mRNA expression of neutrophil-attractant chemokines MIP-2
(CXCL2) and KC (CXCL1) 48 h after LPS administration
in wild-type kidney. Although LPS caused a similar increase in
chemokine expression in ICAM-1⫺/⫺ mice, this increase in
MIP-2 and KC was significantly exaggerated in the CD18-def
group (113 ⫾ 51-, 106 ⫾ 68-, and 330 ⫾ 40-fold increase of
KC in wild-type, ICAM-1⫺/⫺, and CD18-def mice, respectively, P ⬍ 0.05; Table 1). Similarly, LPS strongly increased
mRNA levels of VCAM-1 in wild-type and ICAM-1⫺/⫺ kidney, but this increase was significantly greater in CD18-def
mice (10.3 ⫾ 6.0-, 8.82 ⫾ 4.65-, and 38.8 ⫾ 12.0-fold increase
in wild-type, ICAM-1⫺/⫺, and CD18-def mice, respectively,
P ⫽ 0.09). In support of this, LPS administration increased
VCAM-1 protein expression within kidney in all groups, but to
a significantly greater extent in CD18-def mice compared with
AJP-Renal Physiol • VOL
the others, as assessed by semiquantitative immunohistochemistry scoring (data not shown).
Blockade of adhesion molecules. Given the intact neutrophil
trafficking observed in CD18-deficient mice, the question arose
as to whether residual leukocyte CD18 expression was sufficient to allow adhesion to ICAM-1. Similarly, given its upregulation in CD18-def mouse kidney, the possibility of
VCAM-1-mediated neutrophil infiltration was addressed (14,
51). In wild-type mice, anti-ICAM-1 neutralizing Ab YN1/
1.7.4 successfully blocked LPS-induced ARF in parallel with a
reduction in infiltrating neutrophils (Fig. 7, A-B). In contrast,
anti-ICAM-1 blockade offered no protection against LPSinduced ARF or LPS-induced neutrophilic infiltraton in CD18def mice. Pretreatment with anti-VCAM-1 mAb M/K 2.7 did
not protect against LPS-induced ARF or LPS-induced neutrophilic infiltration in either wild-type or CD18-def mice, if
anything showing a trend toward higher BUN and greater
neutrophil infiltration, although this did not reach statistical
significance (Fig. 7, C-D). Thus neither residual CD18 expression nor VCAM-1 upregulation can explain preserved neutrophil accumulation in CD18-def mice.
DISCUSSION
The ability to direct leukocytes, key effectors of the innate
and adaptive immune response, to the site of inflammation is
governed by a complex network of various chemotactic factors
and adhesion molecules (12, 24). Initially, leukocyte rolling on
endothelium is initiated by interaction between members of the
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Fig. 4. Renal tubular injury following administration of
LPS. LPS caused modest tubular injury in WT mice, most
prominent in the cortex, as manifested by tubular vacuolization and degeneration (A), as well as tubular dilatation
and occasional casts. B: extent of injury was similar in
CD18-def mice, but significantly less in ICAM-1⫺/⫺ mice.
C: semiquantitative cortical tubular injury scores; the score
ranges from 0 for completely normal histology to 9 for
maximal and widespread tubular injury. *P ⬍ 0.05 vs.
CD18-def group, P ⫽ 0.13 vs. WT group. Magnification:
⫻400, periodic acid-Schiff stain.
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ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
selectin family and their carbohydrate ligands. Subsequently, a
transition from rolling to firm adhesion is governed by the
interaction between ␤2-integrins LFA-1 (CD11a/CD18) and/or
Mac-1 (CD11b/CD18) on the leukocyte surface and ICAM-1
expressed on the surface of activated endothelial cells.
ICAM-1 has also been shown to be expressed on other cell
types during inflammation, such as renal tubular cells, which
we confirmed in this study (28). Interference with this receptorligand interaction through a variety of approaches has been
shown both to impair host response to various infections (48),
yet to protect against many injurious effects of the immune
system. Specifically, ICAM-1 has been found to play an
important role in disease models of transplant rejection (36),
radiation-induced pulmonary fibrosis (16), type I hypersensi-
tivity (54), and atherosclerosis (3), among many disease models. It has been previously reported that ICAM-1⫺/⫺ mice are
resistant to LPS-induced mortality (64); although the mechanism of this protection is not known in great detail, the
literature has noted a decrease in LPS-induced neutrophilic
infiltration and organ damage within lung and liver in ICAM1⫺/⫺ mice (1, 20, 30), in lung and eye with use of anti-ICAM-1
neutralizing Abs (2, 30), and in lung and liver with use of an
anti-CD11b neutralizing Ab (43). Similarly, protection against
LPS-induced lung injury was noted in neutrophil-depleted
mice (1). However, until the present study, the role of ICAM1-mediated neutrophil adhesion in LPS-induced ARF has not
been previously examined.
In comparison, much more is known about the role of
leukocyte adhesion in IRI. Interruption of ICAM-1-mediated
neutrophil infiltration has been shown to be protective in
Table 1. Fold increase in RNA expression of selected
chemokines and adhesion molecules as compared with
sham-injected wild-type mice
Fig. 6. Serum TNF levels following LPS administration. At baseline, circulating TNF values were undetectable by sensitive ELISA in all 3 groups of
mice. LPS injection caused a dramatic release of TNF into the circulation in all
mice, peaking at ⬃2 h. However, there were no statistically significant
differences in the extent of TNF release between WT, ICAM-1⫺/⫺, or CD18def mice; n ⫽ 8 per group.
AJP-Renal Physiol • VOL
Group
MIP-2
KC
VCAM-1
Sham
Wild-type
ICAM-1⫺/⫺
CD18-def
1.00⫾0.17*
358⫾210
269⫾170
976⫾252†
1.00⫾0.54*
113⫾51
106⫾68
330⫾40*
1.00⫾0.41*
10.3⫾6.0
8.82⫾4.65
38.8⫾12.0†
Values are means ⫾ SE. MIP-2, macrophage inflammatory protein-2; KC,
keratinocyte-derived chemokine; VCAM-1, vascular cell adhesion molecule-1;
ICAM-1, intercellular adhesion molecule-1. *P ⬍ 0.05 vs. all other groups.
†P ⬍ 0.09 vs. wild-type group and ⬍0.05 vs. other groups.
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Fig. 5. PMN infiltration. A: at baseline, few neutrophils were present within the kidney in all
groups, as seen on frozen kidney sections. B: LPS
administration caused significant neutrophilic infiltration in WT mice at 48 h. C: while similar in
CD18-def mice, neutrophilic infiltration was significantly lower in ICAM-1⫺/⫺ mice. D: average
neutrophils per high-power field (HPF). *P ⬍
0.05 vs. WT and CD18-def groups. Magnification: ⫻400.
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ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
ischemic injury to heart, skeletal muscle, and brain (45, 46,
57). More relevant to the kidney, classic studies by Kelly and
colleagues (22, 23) showed that both ICAM-1⫺/⫺ mice or
wild-type mice given anti-ICAM-1 neutralizing Ab were protected against renal IRI injury, in association with reduced
neutrophil influx. However, a variety of other studies using
neutrophil depletion and other strategies have yielded mixed
results and collectively imply that neutrophils have a relatively
lesser role in this model (47, 49, 58). More recently, the work
of Rabb and colleagues (5) showed in several studies that the
CD4⫹ T cell is more likely to play a central role in renal
ischemic injury. However, significant differences exist between the models of renal IRI and LPS-induced ARF; for
example, the degree of histological damage is much greater in
renal IRI, whereas renal hemodynamic changes and cytokine
release may be more extensive with LPS-induced ARF (37).
While the data described above clearly support a pathogenic
role of ICAM-1 in LPS-induced ARF, these data also show that
the role of the neutrophil is not straightforward. Direct depletion of neutrophils with mAb Gr-1 led to a massive exaggeration in systemic TNF release after LPS and 70% mortality.
This has been reported previously and has been ascribed to the
paracrine action of an uncharacterized low molecular weight
product of neutrophils which inhibits LPS-induced TNF release by macrophages (10, 11). Similarly, a genetic model of
neutropenia has shown exaggerated TNF release in response to
LPS (21). This inhibitory effect toward TNF release does not
seem to involve integrin-ICAM-1 interaction, since TNF release and mortality were not exaggerated in ICAM-1⫺/⫺ or
CD18-def mice, nor in mice receiving anti-ICAM-1 Ab. Neutrophil-depleted mice were not protected against LPS-induced
ARF, and in fact had significantly higher BUN levels 6 h after
AJP-Renal Physiol • VOL
LPS. These data are consistent with a primary role of TNF that
has been previously reported (7, 27), which is in fact greater
than the direct role of LPS itself (9), and clearly indicates that
TNF can cause renal injury through neutrophil-independent
pathways. Possible ways that this may occur include hypotension from massive TNF-mediated induction of inducible nitric
oxide synthase or increased TNFR1-mediated apoptosis. Although this confounding effect of neutrophil depletion makes a
perfectly controlled experiment impossible, low-dose LPS
given to neutrophil-depleted mice led to similar levels of TNF
release (⬃2.5 ng/ml) as seen with high-dose LPS in nonneutrophil-depleted mice and caused no mortality. At this
“appropriate” level of TNF release following LPS, these mice
were protected not only against renal neutrophil infiltration but
also experienced minimal ARF. Taken together, these data
suggest that while they serve to restrain systemic TNF release
in response to LPS, neutrophils themselves may also later play
an injurious role in LPS-induced ARF, given an equivalent
degree of TNF release. A similar dual role of neutrophils was
documented in the cecal ligation and puncture model of sepsis,
where neutrophils are protective and reduce bacteremia early in
the time course but later contribute to organ dysfunction and
mortality (17).
A pathogenic role for neutrophil infiltration is also consistent
with previous work in which interruption of TNFR1 or TLR4
signaling or inhibition of caspases protected against LPSinduced ARF in parallel with a reduction in renal neutrophil
infiltration (7, 9, 15). More directly, hck⫺/⫺fgr⫺/⫺ mice, a
model of impaired neutrophil activation, are protected against
LPS-induced renal dysfunction (32). It should be noted that
ICAM-1⫺/⫺ mice were only partially protected against LPSinduced ARF, so it is likely that other mechanisms, such as
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Fig. 7. Influence of ICAM-1 and vascular cell
adhesion molecule-1 (VCAM-1) blockade on
LPS-induced ARF and neutrophil infiltration.
WT control mice receiving LPS developed
ARF, as expected. However, WT mice pretreated with anti-ICAM-1 mAb YN1/1.7.4
were protected, both in terms of BUN (A) and
renal neutrophil accumulation (B). CD18-def
mice pretreated with anti-ICAM-1 mAb were
not protected. Blockade of VCAM-1 with
mAb M/K-2.7 did not protect either WT or
CD18-def mice against LPS-induced ARF or
neutrophil infiltration (C-D) compared with
rat-IgG-injected control mice. *P ⫽ 0.05 vs.
IgG group, *P ⫽ 0.07 vs. CD18-def group.
**P ⬍ 0.01 vs. both other groups; n ⫽ 6 per
group.
ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
AJP-Renal Physiol • VOL
ade of ICAM-1 in CD18-def mice did not block neutrophil
influx. Because ICAM-1 has a short intracellular sequence
lacking intrinsic kinase activity or Src homology domains, a
signaling role for ICAM-1 independent of CD18 is less likely,
although some authors have ascribed roles for ICAM-1 in
processes such as angiogenesis or glutathione metabolism (25,
26). Another possibility is that ICAM-1 could mediate injury
independent of leukocytes through binding of its ligand fibrinogen (31, 50). Such a role for ICAM-1 independent of neutrophil recruitment would still raise the question of what factors
are responsible for neutrophil recruitment to the kidney, a
process normally attributed to adhesion molecules and chemokines.
Alternatively, as with any genetically altered or mutant
animal, compensatory mechanisms may have developed to
compensate for the absence of normal CD18 expression. This
might be expected to be more likely with CD18 deficiency, as
opposed to ICAM-1 deficiency, since unlike ICAM-1, CD18 is
expressed at fairly high levels at baseline, before an inflammatory stimuli (39). Multiple defects in adaptive immunity and
in trafficking of various leukocyte types have been observed in
CD18-deficient mice (33, 35). Other investigators described
compensatory changes that preserve leukocyte migration or
adhesion in genetically altered strains (13, 48, 61). As a
possible example of this, we noted exaggerated expression of
VCAM-1 and neutrophil-attracting chemokines MIP-2 and KC
in response to LPS in CD18-def mice compared with wild-type
mice. Since blockade of VCAM-1 with mAb M/K-2.7 did not
protect CD18-def mice against LPS-induced neutrophil infiltration or ARF, VCAM-1 upregulation is not responsible for
this preserved neutrophil infiltration. Therefore, increased synthesis of MIP-2 and KC may be a more likely explanation. It
should also be noted that although we observed no exaggeration in LPS-induced TNF release in CD18-def mice, others
have noted a greater intrinsic innate immune response in this
strain (10).
In conclusion, these data support both an important protective role for neutrophils in inhibiting LPS-induced TNF release
yet an adverse role for ICAM-1 in LPS-induced ARF. It should
be noted that this LPS model is a rather simplified simulation
of clinical sepsis in humans. In addition to LPS, a variety of
bacterial products, such as fMLP and unmethylated bacterial
DNA, may directly activate the innate immune system over a
chronic or intermittent time course. To add to this complexity,
clinical sepsis often occurs in a population with substantial
comorbidities, yet in the setting of various supportive measures, such as hemodynamic drugs, mechanical ventilation, and
antibiotics. Other investigators (37) used models such as cecal
ligation and puncture in fluid-resuscitated aged mice to simulate septic ARF in humans, which could potentially demonstrate different results for the role of neutrophils and ICAM-1.
It is hoped that greater insight into this process of leukocyte
dynamics in sepsis may eventually lead to therapeutic strategies aimed at treatment and prevention of ARF. This may
prove challenging, as neutrophils and other leukocytes clearly
have an important role in combating the microorganisms responsible for sepsis; an anti-adhesion molecule strategy would
be expected to have fewer adverse consequences in ARF
associated with syndromes of sterile inflammation, such as
ARF in the setting of pure ischemia or postsurgical states.
Alternatively, greater understanding of the exact ways in which
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hemodynamic changes or damage from reactive oxygen species, remain relevant in this model. Additionally, ICAM-1⫺/⫺
mice and mice receiving anti-ICAM-1 mAb still had some
infiltration of the kidney with neutrophils after LPS injection,
although to a significantly lesser extent than wild-type mice;
thus other adhesion molecules, for example members of the
selectin family, still allow for some leukocyte trafficking
within the kidney in the setting of widespread inflammation.
This is consistent with multiple other studies that have implicated leukocyte adhesion pathways independent of the CD18/
ICAM-1 interaction and which have demonstrated that mediators of chemotaxis differ somewhat between different tissues
(4, 40). Deficiency of ICAM-1 may have conferred protection
against LPS-induced ARF not only by decreasing neutrophil
adhesion, but also by abrogating signaling events that occur in
endothelium upon ligation of ICAM-1 (6, 60), or possibly
through other as yet unknown neutrophil-independent mechanisms. Last, ICAM-1 deficiency did not confer protection by
reducing other mediators of inflammation, such as LPS-induced TNF release or renal chemokine expression. This is
consistent with the understanding that ICAM-1 is one of
several downstream effectors induced by TNF (44). Consistent
with this, the modest degree of protection we described in
ICAM-1⫺/⫺ mice is much less than the degree of protection
seen in TNFR1⫺/⫺ mice (7).
Infiltration of the kidney with neutrophils or other leukocytes could lead to ARF in several different ways. Neutrophils
attached to vessel walls could lead to reduced renal blood flow,
as has been suggested by intravital microscopy studies (41, 42).
This could happen from either direct physical obstruction of
small peritubular capillaries, or possibly from damage to endothelial cells from the local release of reactive oxygen species, proteases, or cytokines, which could in turn trigger
endothelial sloughing, vasoconstriction, or procoagulant effects. Depending on the extent to which neutrophils escape the
vasculature and come in to contact with renal tubules, release
of these injurious mediators could also result in direct tubular
damage and consequent decrease in renal function (63). The
experiments described above do not directly address which of
these mechanisms are most relevant, so answering this question will require further study. We did observe that neutrophilic
infiltration was associated with increased tubular injury, which
may have been due either to direct damage from infiltrating
leukocytes and/or ischemia from upstream vascular obstruction.
Since the principal ligands for ICAM-1 are CD11a/CD18
and CD11b/CD18, we did not anticipate the lack of protection
against LPS-induced ARF in CD18-def mice. In contrast to our
results showing preserved neutrophil infiltration within the
kidney, this strain of CD18-def mice does exhibit reduced
neutrophil chemotaxis in chemical peritonitis (62), similar to
ICAM-1⫺/⫺ mice (55). The fact that CD18-def mice still
experienced robust neutrophil infiltration in response to LPS,
in parallel with functional and structural kidney injury, remains
consistent with a role for neutrophil-mediated kidney injury in
LPS-induced ARF. It should be noted that this strain of mice
used in our studies still expresses CD18 on circulating leukocytes at levels ⬃2% of wild-type, which may increase to 16%
under inflammatory stimuli (62). However, interaction between
residual CD11a/CD18 or CD11b/CD18 and ICAM-1 cannot
explain preserved neutrophil recruitment after LPS, as block-
F1269
F1270
ICAM-1 IN LPS-INDUCED ACUTE RENAL FAILURE
adhesion molecules contribute to acute renal dysfunction could
yield more specific and targeted therapies.
ACKNOWLEDGMENTS
We thank B. Hack and Dr. Y. Nakagawa for technical assistance.
GRANTS
This work was supported by National Institutes of Health Grant K08-DK61375.
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