Resistin Inhibits Essential Functions of
Polymorphonuclear Leukocytes
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J Immunol 2008; 181:3761-3768; ;
doi: 10.4049/jimmunol.181.6.3761
http://www.jimmunol.org/content/181/6/3761
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References
Gerald Cohen, Dalibor Ilic, Jana Raupachova and Walter H.
Hörl
The Journal of Immunology
Resistin Inhibits Essential Functions of Polymorphonuclear
Leukocytes
Gerald Cohen,1 Dalibor Ilic, Jana Raupachova, and Walter H. Hörl
P
olymorphonuclear leukocytes (PMNLs)2 are cells of the
first-line nonspecific immune response. PMNLs from patients with chronic kidney disease (CKD) are characterized by an increased basal activity and a diminished reactivity
upon stimulation leading to an increased incidence of morbidity
and mortality (1–3). The former feature contributes to the inflammatory state described in uremic patients and as a consequence to
an increased risk for cardiovascular diseases. The diminished reactivity of PMNLs is related to the increased occurrence of bacterial infections in uremia. Uremic toxins, molecules that accumulate in the sera of uremic patients as a result of kidney failure,
contribute to a complex mixture of organ dysfunctions (4). Various
uremic retention solutes which contribute to the disturbed immune
function in uremia have been isolated and characterized (5–9).
The serum concentrations of the “adipocytokines” leptin and
resistin are elevated in patients with end-stage renal disease (10).
Leptin, originally described as a signal of satiety to the brain, is
also involved in the regulation of immune and inflammatory processes (11). A few reports on the influence of leptin on PMNLs
have been published previously (12–15). Resistin, a protein secreted by mouse adipocytes, was suggested to be associated with
insulin resistance (16). However, in contrast to rodents, resistin is
Division of Nephrology and Dialysis, Department of Medicine III, Medical University of Vienna, Vienna, Austria
Received for publication August 20, 2007. Accepted for publication July 7, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
Address correspondence and reprint requests to Dr. Gerald Cohen, Medizinische
Universität Wien, Univ.-Klinik für Innere Medizin III, Abteilung für Nephrologie und
Dialyse, Währinger Gürtel 18 –20, Vienna A-1090, Austria. E-mail address:
[email protected]
2
Abbreviations used in this paper: PMNL, polymorphonuclear leukocyte; CKD,
chronic kidney disease; HD, hemodialysis; GSK-3, glycogen synthase kinase 3.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
www.jimmunol.org
expressed primarily in inflammatory cells in humans, especially in
macrophages (17, 18), and resistin mRNA was readily detected in
circulating human mononuclear cells (19). Resistin serum levels
are also increased in patients with type 2 diabetes mellitus (20, 21),
which have an increased risk of infectious episodes as a result of
impaired PMNL functions (22, 23). Resistin has a positive effect
on endothelial cell activation (24), but there are–to our knowledge–no reports on the effect of resistin on PMNL functions. The
goal of this study was to assess the effect of resistin on essential
PMNL functions such as chemotaxis, oxidative burst, glucose uptake, and phagocytosis and thereby shed light on the contribution
of resistin to a disturbed immune response.
Materials and Methods
Materials
Human recombinant resistin was purchased from BioVendor Laboratory
Medicine. Ficoll-Hypaque and HBSS were obtained from Amersham Biosciences and Invitrogen, respectively. Saline solution (0.9% NaCl) was
purchased from Fresenius Kabi, and Giemsa stain was obtained from
Merck. Polystyrene conical tubes and culture tissue dishes used for chemotaxis assays were purchased from BD Discovery Labware, and Vacutainer tubes for blood collecting (Vacuette) were obtained from Greiner
Bio-One. All other chemicals were purchased from Sigma-Aldrich, unless
otherwise indicated.
Resistin serum concentrations
Serum concentrations of resistin in healthy subjects and hemodialysis (HD)
patients were determined using the quantitative sandwich enzyme immunoassay technique. The commercially available human resistin immunoassay (Quantikine) was purchased from R&D Systems. The microtiter plates
were precoated with a mAb specific for resistin. Resistin in the serum
sample was bound to the immobilized Ab. Unbound substances were
washed away, and a mouse mAb specific for resistin conjugated to HRP
was added. After washing away any unbound reagent, a substrate solution
(tetramethylbezidine) was added. The color development was stopped by
adding sulfuric acid, and the OD was measured by a microplate reader
(Anthos Reader HT 3; Anthos Labtec Instruments) at 450 nm.
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The serum levels of resistin, a 12-kDa protein primarily expressed in inflammatory cells in humans, are increased in patients with
chronic kidney disease and in those with diabetes mellitus. Both groups of patients have an increased risk of infections mainly as
a result of disturbed polymorphonuclear leukocyte (PMNL) functions. Therefore, we investigated the influence of resistin on
human PMNLs. Serum resistin concentrations were determined with a sandwich enzyme immunoassay. Using PMNLs from
healthy subjects, chemotaxis was tested by the under-agarose method. Flow cytometric assays to measure oxidative burst and
phagocytosis were conducted in whole blood. The uptake of deoxyglucose was determined as measure of the PMNL activation
state. The activity of intracellular kinases was assessed by Western blotting and by in vitro kinase assays. Resistin inhibited PMNL
chemotaxis and decreased the oxidative burst stimulated by Escherichia coli and by PMA, but did not influence PMNL phagocytosis of opsonized E. coli and PMNL glucose uptake. The inhibition of PMNLs by resistin was observed at concentrations found
in serum samples of uremic patients, but not in concentrations measured in healthy subjects. Experiments with specific signal
transduction inhibitors and measurements of intracellular kinases suggest that PI3K is a major target of resistin. In conclusion,
resistin interferes with the chemotactic movement and the stimulation of the oxidative burst of PMNL, and therefore may
contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic
subjects. The Journal of Immunology, 2008, 181: 3761–3768.
3762
Isolation of PMNLs
PMNLs were isolated from heparinized venous blood of healthy volunteers
by using discontinuous Ficoll-Hypaque (Amersham Biosciences) density
gradient centrifugation and hypotonic lysis of erythrocytes as previously
described (7). Informed consent was given by all donors. The donors for
leukocyte function analysis were 33.4 ⫾ 1.2 years old (mean value ⫾
SEM; n ⫽ 52) and consisted of the same numbers of male and female
volunteers with normal C-reactive protein, creatinine, and normal fasting
blood glucose levels. Smokers, people taking medication or having infection or inflammation, and diabetic subjects were excluded. Ten ml of venous blood was collected in sterile lithium heparin Vacutainer tubes, mixed
with 10 ml 0.9% saline, and then layered on top of 12 ml of FicollHypaque. The mixture was centrifuged (20°C, 30 min, 450 g) and erythrocytes were removed from the pellet by hypotonic lysis with ammonium
chloride buffer (157 mM NH4Cl, 10 mM KHCO3; Merck; and 0.1 mM
EDTA; Sigma-Aldrich) on ice. The PMNLs were collected by centrifugation (4°C, 10 min, 250 g) and washed in the buffer used in the following
test. The cells were counted in a cell counter (CellDyn610; Abbott Laboratories). The viability of the PMNLs obtained by this protocol was ⬎95%
as determined by the exclusion of ethidium bromide (Invitrogen) under the
fluorescence microscope.
Chemotaxis
Oxidative burst
The quantitative determination of the PMNL oxidative burst was conducted after adding 10 ␮l of resistin stock solutions or 10 ␮l PBS to 90 ␮l
heparinized whole blood. The oxidative burst was stimulated by unlabeled
opsonized Escherichia coli or by PMA. Dihydrorhodamine 123 was used
as fluorogenic substrate (Bursttest; Opregen Pharma). The evaluation of the
oxidative burst activity, the conversion of dihydrorhodamine 123 to rhodamine 123 by reactive oxygen species produced by PMNLs, was performed by flow cytometry (Epics XL-MCL; Beckman Coulter). The mean
fluorescence intensity of the unstimulated sample in the absence of resistin
was set as 100%.
Inhibition experiments
The signal transduction inhibitors SB203580, PD98059, and LY294002 at
final concentrations of 30, 50, and 10 ␮M, respectively, were added to the
assay. These inhibitors were purchased from Calbiochem. Because the
compounds had to be dissolved in DMSO to prepare a stock solution, the
end concentration of DMSO was 0.1% (v/v). DMSO at this concentration
had no effect on the results and was used as control.
Activity of intracellular kinases: Western blot and in vitro
kinase assay
Incubations. PMNLs (3 ⫻ 106) in 100 ␮l were incubated at 37°C for 5
min in the presence of resistin at final concentrations of 70 ng/ml and 25
ng/ml, respectively, or in the presence of PBS alone. Then, to the cells 20
␮l of PBS (basal value) or 20 ␮l stimulant solution were added and incubated for another 5 min. The final concentration for fMLP was 1 ⫻ 10⫺7M,
for PMA 1.35 ␮M, and for E. coli (taken from the Bursttest kit, see above)
1.2 to 2.4 ⫻ 108 cells per ml.
Preparation of extracts. The cells were centrifuged (4°C, 4 min, 590 ⫻ g),
washed twice in ice-cold PBS, and taken up in 125 ␮l lysis buffer (20 mM
Tris (pH7.5); 150 mM NaCl; 1 mM EDTA; 1 mM EGTA, 1%(v/v) Triton-
X100; 2.5 mM sodium pyrophosphate; 1 mM Na3VO4; 1 ␮g/ml leupeptin;
1 mM phenyl-methyl sulfonyl fluoride). After addition of 6.25 ␮l protease
inhibitor mixture, the cells were vortexed for 30 s, left on ice for 5 min, and
sonicated 4 times for 5 s each. Then the extracts were centrifuged (4°C,
10 s, 10,000 ⫻ g). A total of 15 ␮l of the supernatant were directly used
for Western blotting and 100 ␮l for the in vitro kinase assay.
In vitro kinase assay. The activity of Akt (also referred to as protein kinase
B), the downstream effector of PI3K, was determined with the nonradioactive Akt Kinase Assay kit (Cell Signaling Technology). A total of 10 ␮l
of immobilized anti-Akt Ab bead slurry was added to 100 ␮l cell lysate and
incubated over night at 4°C. After centrifugation (4°C, 30 s, 14,000 ⫻ g)
the beads were washed twice in lysis buffer and twice in kinase buffer (2.5
mM Tris (pH7.5); 500 ␮M ␤-glycerolphosphate, 200 ␮M DTT; 10 ␮M
Na3VO4, 1 mM MgCl2), 250 ␮l each. The pellet was suspended in 10 ␮l
kinase buffer supplemented with 200 ␮M ATP and 0.2 ␮l kinase substrate,
i.e., glycogen synthase kinase 3 (GSK-3) fusion protein, and incubated at
30°C for 30 min.
Western blotting. To the PMNL lysate or the sample after the in vitro
kinase reaction, one-half volume of 3⫻ sample buffer (187mM Tris (pH
6.8); 6% (v/v) SDS; 0.03% (w/v) bromphenol blue; 7.5% (v/v) 2-ME) was
added and heated to 95°C for 3 min. After separating the proteins by
SDS-PAGE (PhastSystem; Amersham Biosciences), they were electrotransferred to a nitrocellulose membrane. The primary Abs were polyclonal
rabbit anti-p38 MAPK, anti-phospho-p38 MAPK, anti-p44/42 kinase, antiphospho-p44/42 kinase, and anti-phospho-GSK-3 Abs (Cell Signaling
Technology). They were detected by HRP- labeled goat-anti-mouse Abs,
and the ECL detection system (Amersham Biosciences) using Hyperfilm
ECL (Amersham Biosciences). The intensity of the bands was determined
by scanning with the Personal Densitometer SI (Molecular Dynamics) using the ImageMaster 1D software (Amersham Biosciences). The films were
exposed to the blots for different times, and bands within the linear range
were chosen for evaluation.
Glucose uptake
The hexose uptake was assessed as previously described (9). Isolated
PMNLs were resuspended in PBS at a concentration of 1 ⫻ 106 cells/ml
and preincubated for 5 min at 37°C in a shaking water bath. Then up to 20
␮l of PBS containing resistin stock solution or the same volume of PBS
were added to a 200-␮l cell suspension and incubated for 15 min. Ten ␮l
of PBS or fMLP at a final concentration of 10⫺7 M were added and the
incubation was continued for 15 min at 37°C. Then 100 ␮l of 2-deoxy-D[1-3H]glucose (PerkinElmer) stock solution (5 ␮Ci per ml of 100 ␮M
D-glucose in PBS) were added and incubated for 60 min. The uptake was
stopped by adding 1 ml ice-cold PBS. The samples were centrifuged at
20,000 ⫻ g for 20 s, and the pellet was washed once with ice-cold PBS.
Afterward the supernatant was removed and the pellet together with the 1.5
ml-reaction tube was put into a scintillation vial containing 10 ml Optifluor
(Packard Instrument B.V.; Chemical Operations). The incorporated radioactivity was determined with a liquid scintillation counter.
Phagocytosis
The phagocytotic activity of PMNLs was quantified in heparinized whole
blood. Flow cytometry (Epics XL-MCL) was used to determine the percentage of PMNL that had taken up FITC-labeled opsonized E. coli
(Phagotest; Opregen Pharma) and the amount of ingested E. coli per PMNL
in the absence and presence of resistin.
Statistical analysis
Wilcoxon matched-pair signed-rank statistic was used for data analysis of
assays testing the PMNL functions influenced by resistin. Differences in
resistin serum levels were assessed with the t test. Data are presented as
mean values ⫾ SEM.
Results
Resistin serum concentrations in healthy subjects and HD
patients
The serum resistin level of healthy subjects was 12.8 ⫾ 1.2 ng/ml.
The values were significantly higher in females as compared with
males (Table I). The resistin concentrations in sera of HD patients
were significantly elevated as compared with healthy subjects
(33.0 ⫾ 2.7 ng/ml). However, in contrast to healthy subjects, male
HD patients had significantly higher serum resistin levels than female patients (Table I). In the light of the gender differences regarding the serum resistin concentrations in healthy subjects, it is
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PMNL chemotaxis was determined by the under-agarose method as previously described (7). PMNLs were resuspended at a concentration of
0.5 ⫻ 106/10 ␮l in PBS (pH 7.2; Invitrogen) or in PBS containing resistin
and incubated for 15 min at 37°C before the start of the chemotaxis. fMLP
in Hank’s buffer (HBSS; Invitrogen) was used as a chemoattractant at a
final concentration of 4.2 ⫻ 10⫺7 M. During the migration step the agarose
plates were incubated for 2 h at 37°C. Afterward, the PMNLs were fixed
with methanol and paraformaldehyde and stained with Giemsa (Merck).
The distance the cells migrated under the agarose was measured under the
microscope.
To assess the reversibility of the effects of resistin on PMNL chemotaxis, the cell suspension was incubated in the presence of resistin for 30
min at 37°C. Then the PMNLs were washed twice, resuspended in PBS,
and applied to the chemotaxis assay. Because centrifugation and suspending the cells could interfere with the results, the washed PMNLs were
compared with cells that underwent the same incubation-centrifugationresuspension protocol, except that the supernatant was not removed after
centrifugation and the cells were resuspended in the resistin solution
(supernatant).
RESISTIN INHIBITS PMNL FUNCTIONS
The Journal of Immunology
3763
Table I. Resistin concentrations in sera of healthy subjects (HS) and hemodialysis patients (HD) as
determined by ELISA (mean values ⫾ SEM)
Total
Male
Female
HS
12.8 ⫾ 1.2 (n ⫽ 30) 10.1 ⫾ 0.7 (n ⫽ 15) 15.4 ⫾ 1.4 (n ⫽ 15)
HD
33.0 ⫾ 2.7 (n ⫽ 27) 40.5 ⫾ 2.8 (n ⫽ 14) 24.9 ⫾ 1.5 (n ⫽ 13)
p Value (HS vs HD)
2.1 ⫻ 10⫺7
3.0 ⫻ 10⫺7
3.7 ⫻ 10⫺3
important to state that the response of PMNLs obtained from
healthy donors was comparable between males and females in all
functional assays performed (see below).
Effect of resistin on PMNL chemotaxis
Effect of resistin on PMNL oxidative burst
The oxidative burst assay was performed in whole blood of healthy
subjects. We increased the in vivo resistin concentration by the
addition of 12.5, 25, and 100 ng/ml. An increase by 12.5 ng/ml
already lead to a significant reduction of the stimulation of the
oxidative burst induced by E. coli (Fig. 3A). Higher resistin concentrations did not lead to further inhibition. Resistin also reduced
the stimulation of the oxidative burst by PMA (Fig. 3B). In this
case, the resistin concentration had to be brought 25 ng/ml above
the normal value to observe a significant inhibitory effect. There-
FIGURE 1. Effect of resistin on PMNL chemotaxis. Migration of
PMNLs toward fMLP in the absence (0) or in the presence of 25 ng/ml
resistin (R), or after preincubation with 25 ng/ml resistin and subsequent
removal by washing the cells (R r). n ⫽ 6. Mean values ⫾ SEM. ⴱ, p ⬍
0.05 vs 0 ng/ml. $, p ⬍ 0.05 vs R.
0.037
0.003
fore, uremic resistin levels attenuate the stimulation of the oxidative burst as compared with normal levels, i.e., conditions where
no resistin was added to the blood.
In a next set of experiments, we tested the effect of the signal
transduction inhibitors SB203580, PD98059, and LY294002 on
the activation of the oxidative burst in the absence and presence of
resistin. Fig. 4, A and B shows that all inhibitors alone significantly
inhibited the activation of the oxidative burst by both E. coli and
PMA, except that PD98059 did not reduce the stimulation by E.
coli. The strongest effect was observed when PI3K was inhibited
by LY294002 before the stimulation by PMA. This suggests that
ERK (p44/42) is not involved in the E. coli stimulated burst,
whereas PI3K seems to play a major role when PMA is used as a
stimulant. In contrast to the stimulation of the oxidative burst by
PMA (Fig. 4B), resistin had a strong additive effect to the inhibition of the E. coli stimulated burst by SB203580 and LY294002
(Fig. 4A).
Activation of intracellular kinases
Fig. 5, A–C shows that fMLP, E. coli, and PMA induce a significant phosphorylation and thereby activation of p38 MAPK,
whereas these stimuli did not affect the expression levels of total
p38 MAPK. The presence of resistin did not lead to a change on
the phosphorylation status of stimulated or unstimulated cells. Experiments assessing the activation levels of ERK (p44/42) in the
absence or presence of resistin showed similar results as for p38
MAPK (Fig. 6, A–C). Fig. 7 shows that both fMLP and PMA lead
to a significant increase in PI3K/Akt activity. Whereas resistin had
FIGURE 2. Effect of signal transduction inhibitors on resistin-attenuated PMNL chemotaxis. Migration of PMNLs toward fMLP in the absence
(gray bars) or in the presence (black bars) of 25 ng/ml resistin and the
inhibitors SB203580 (SB; 30 ␮M), PD98059 (PD; 50 ␮M), and LY294002
(LY; 10 ␮M). Co, Control in the absence of inhibitors. Six to eight independent experiments in the presence of inhibitors were performed. Mean
values ⫾ SEM. ⴱ, p ⬍ 0.05 vs control in the absence of resistin; $, p ⬍ 0.05
vs the absence (gray bars) of resistin.
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Resistin significantly attenuated PMNL chemotaxis at a concentration found in uremia (25 ng/ml; Ref. 10; Fig. 1). When the
chemotaxis assay was performed with PMNL preincubated with
resistin and subsequent removal of the protein, an attenuated inhibition of chemotaxis was observed which was significantly lower
than in the presence of resistin. Hence, the resistin effect on PMNL
chemotaxis was partially reversible (Fig. 1).
We tested the influence of SB203580 (an inhibitor of p38
MAPK), of PD98059 (an inhibitor of ERK, p44/42), and of
LY294002 (an inhibitor of PI3K) on PMNL chemotaxis in the
absence and presence of resistin. Fig. 2 shows that all inhibitors
alone exerted an inhibitory effect on PMNL chemotaxis, indicating
that p38 MAPK, ERK, and PI3K are involved in the regulation of
the chemotactic movement of PMNLs toward fMLP. Resistin did
not show an additional augmentation of inhibition (Fig. 2).
p Value
(Male vs Female)
3764
no effect on PI3K/Akt basal activity, it abolishes both fMLP- and
PMA-induced activation, in the case of PMA even below basal
levels. These data suggest that PI3K is the major target of resistin
among the three kinases tested in this study.
Furthermore, we found that the presence of E. coli induced a
decrease of PI3K/Akt activity in the absence and presence of resistin (Fig. 7). We found that this is the result of phosphatases
activated during the incubation of PMNLs with E. coli (unpublished observation).
Effects of resistin on PMNL glucose uptake
We investigated the effects of resistin on the uptake of 3H-labeled
2-deoxy-D-glucose by isolated PMNLs, considered as a measurement of the metabolic activity these cells. The activation of the
glucose uptake by fMLP was not influenced by resistin at final
concentrations of 12.5 ng/ml, 25 ng/ml, and 100 ng/ml (data not
shown).
Effects of resistin on PMNL phagocytosis
The percentage of PMNLs taking up opsonized E. coli bacteria in
whole blood was 88 ⫾ 3% as determined by an assay based on
FIGURE 4. Effect of signal transduction inhibitors on the resistin-attenuated oxidative burst of PMNLs. PMNLs were stimulated by E. coli (A) or
by PMA (B) in the absence (gray bars) or in the presence (black bars) of
25 ng/ml resistin and the inhibitors SB203580 (SB; 30 ␮M), PD98059 (PD;
50 ␮M), and LY294002 (LY; 10 ␮M). Co: control in the absence of inhibitors. Six to 10 independent experiments in the presence of inhibitors
were performed. Mean values ⫾ SEM. ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.01 vs
control in the absence of resistin; $, p ⬍ 0.05 and $$, p ⬍ 0.01 vs the
absence (gray bars) of resistin.
flow cytometry. This value did not significantly change when the
resistin concentration of was increased (data not shown). The number of E. coli taken up per PMNL was not changed either in the
presence of higher resistin concentrations (data not shown).
Discussion
Resistin is a 12.5-kDa protein secreted by mouse adipocytes. In
rodents, adipose tissue is the main source of resistin and is considered as a link between obesity and diabetes by impairing insulin
sensitivity and glucose tolerance (16). In humans, however, resistin is expressed primarily in macrophages (17) and represents a
potential novel link between inflammation and adipocytokines
(25). Its role in human insulin resistance remains controversial.
Whereas some groups (26 –28) describe an association between
serum resistin levels and insulin resistance in humans, others (29 –
31) did not find increased resistin concentrations in insulin resistant patients.
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FIGURE 3. Effect of resistin on PMNL oxidative burst. PMNLs were
stimulated by E. coli (A) or by PMA (B). Resistin was added to whole
blood and thereby the resistin concentration increased by 12.5, 25, and 100
ng/ml. Six to eight independent experiments were performed. Mean values ⫾ SEM. ⴱ, p ⬍ 0.05 vs 0 ng/ml.
RESISTIN INHIBITS PMNL FUNCTIONS
The Journal of Immunology
3765
The level of resistin is elevated not only in the blood of patients
with type 2 diabetes mellitus (20, 21), but also in those with CKD
(10, 29). Therefore, resistin is a potential uremic toxin contributing
to the disturbed immune response in uremic patients by affecting
functional abilities of PMNLs, cells which play a key role in the
nonspecific immune defense against bacterial infections. Increased
serum resistin concentrations in HD patients may be a result of
CKD and/or inflammation (25), considering that human resistin is
primarily expressed by inflammatory cells and that resistin levels
are positively associated with levels of inflammatory markers
(32–34).
Resistin levels used in this study were within the range measured in the sera of healthy subjects and of HD patients (Table I).
The highest resistin concentration used for the in vitro experiments
in this work was 100 ng/ml.
In healthy subjects the values were significantly higher in females than in males (15.4 ⫾ 1.4 ng/ml vs 10.1 ⫾ 0.7 ng/ml, p ⫽
0.037). A previously published study (35) analyzing the serum
resistin levels of adolescents with a mean age of 17.7 ⫾ 1.8 years
found levels of 16.9 ⫾ 5.4 ng/ml in females and 14.0 ⫾ 7.2 ng/ml
in males ( p ⬍ 0.02), whereas Malyszko et al. measured similar
resistin levels in men and women (25). Of note, in contrast to
healthy subjects male HD patients had significantly higher serum
resistin levels than female patients (Table I). At present, an explanation for this difference cannot be provided.
The chemotactic movement of PMNLs toward sites of infection
and injury is an early essential step of the nonspecific immune
response. In the present work, we show that resistin at uremic
concentrations significantly attenuates PMNL chemotaxis (Fig. 1).
The observed effect may be relatively small. However, resistin is
one of several uremic retention solutes contributing to the diminished chemotaxis in CKD patients and we previously showed that
a combination of uremic toxins may attenuate PMNL chemotactic
movement in a synergistic manner (7).
The inhibitory effect of resistin on PMNL migration was partially reversible (Fig. 1) suggesting that lowering the serum resistin
concentration (e.g., by dialysis treatment) might improve PMNL
chemotactic activity. However, preliminary data from our group
(unpublished observation) and a study by Nusken et al. (36) show
that HD is not able to lower serum resistin levels in uremic patients. Of note, a temporary incubation of PMNLs with a previously described chemotaxis-inhibiting protein was sufficient for a
nonreversible inhibition of cell migration (6).
We tested the effect of resistin on the activation of the oxidative
burst by opsonized E. coli bacteria and by PMA, a direct activator
of protein-kinase C. Addition of 12.5 ng/ml resistin to whole blood
samples results in an in vitro concentration found in uremia. Therefore, the data in Fig. 3A show that a uremic resistin concentration
(12.5 ng/ml added and above) significantly inhibited the stimulation of the oxidative burst by E. coli as compared with normal
serum levels (no resistin added to whole blood samples). The resistin concentration had to be brought 25 ng/ml above normal to
observe a significant inhibitory effect of PMA stimulation (Fig.
3B).
PMNLs of patients with diabetes mellitus show decreased chemotaxis (22, 23) and a reduced oxidative burst stimulated with
PMA (37, 38) and opsonized zymosan (37). Therefore, our data
suggest that increased resistin serum levels may contribute to the
disturbed PMNL responses in this group of patients as well.
The effect of resistin on the uptake of glucose by a variety of cell
types has previously been studied. The insulin-stimulated glucose
uptake by mouse adipocytes is enhanced by neutralization of resistin (16). Recombinant resistin inhibited insulin-stimulated glucose uptake by rat skeletal muscle cells (39). Chronic treatment of
differentiated human preadipocytes with recombinant resistin reduced glucose uptake (21), and murine as well as human native
resistin reduced insulin-stimulated glucose uptake in isolated
mouse cardiomyocytes (40). In this study, we found that human
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FIGURE 5. Effect of resistin on p38 MAPK activation. Western blots of extracts of PMNLs incubated in the presence of 25 ng/ml resistin or buffer alone.
Detection of p38 MAPK (p38) and phospo-p38 MAPK (P-p38). n ⫽ 5 to 7. PMNL were unstimulated or stimulated by fMLP (A), E. coli (B), or PMA
(C). Representative Western blots and the densitometry evaluation of the blots are shown. Mean values ⫾ SEM. ⴱ, p ⬍ 0.05 vs unstimulated cells in the
absence of resistin.
3766
RESISTIN INHIBITS PMNL FUNCTIONS
recombinant resistin had no effect on the glucose uptake by
PMNLs isolated from healthy subjects.
In agreement with the findings of Caldefie-Chezet et al. (13),
resistin did not change PMNL phagocytosis in our experiments.
Increases in the cytosolic Ca2⫹ concentration modulate various
PMNL functions (41). However, preliminary experiments in our
laboratory showed that resistin does not affect the basal or fMLPstimulated
cytosolic
Ca2⫹
concentration
(unpublished
observation).
Resistin attenuates multiple effects of insulin, including PI3K
activation and activation of Akt in mouse adipocytes (42). The
MAPK and PI3K pathways are involved in the signal transduction
in several PMNL effector functions (43). Therefore, we investigated the involvement of the MAP kinases ERK (p44/42) and p38
MAPK and of PI3K in the resistin effects described above by using
specific inhibitors. Our inhibition experiments suggest that p38
MAPK, ERK (p44/42) as well as PI3K are involved in the regulation of PMNL chemotaxis toward fMLP (Fig. 2). Consistent with
our findings, Azuma et al. (44) showed that SB203580 attenuates
PMNL chemotaxis and that fMLP stimulates p38 MAPK via mul-
tiple pathways. However, p38 MAPK is not sufficient to induce
chemotaxis (45), explaining the rather small effect of the inhibitor.
The involvement of ERK (p44/42) in PMNL chemotaxis is contradictory. Whereas Zu et al. (46) did not observe an effect of the
ERK inhibitor PD98059 on PMNL chemotaxis, Coxon et al. (47)
showed that inhibition of ERK attenuates chemotaxis. Furthermore, Nagata et al. (48) found that PD98059 inhibits fMLP-stimulated chemotaxis and Chang et al. (49) showed that fMLP activates ERK via PLC in PMNL. PI3K was found to be pivotal for
leukocyte chemotaxis (50). However, Heit et al. (51) demonstrated
that PI3K enhances early responses to fMLP, but is not required
for migration. This may explain the small and partial– even though
significant– effect observed in our assay. The presence of resistin
had no significant influence on the effect of the inhibitors. To identify potential targets of the resistin inhibitory effect, we performed
FIGURE 7. Effect of resistin on the stimulation of Akt activity. Relative
Akt-activity in the absence (0) or in the presence of 25 ng/ml resistin (R)
in unstimulated PMNLs (0) or stimulated by fMLP (F), E. coli (E), or PMA
(P). Phospho-GSK3 (P-GSK3) was detected by Western blotting.
FIGURE 8. fMLP-induced PMNL chemotaxis. Simplified pathways
showing the kinases involved, the corresponding inhibitors (SB203580:
SB; PD98059: PD; LY294002: LY), and the suggested target of resistin.
MEK1,2, MAPK kinases.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 6. Effect of resistin on p44/42 MAPK activation. Western blots of extracts of PMNLs incubated in the presence of 25 ng/ml resistin or buffer
alone. Detection of p44/42 MAPK (p44/42) and phospho- p44/42 MAPK (P-p44/42). n ⫽ 4 to 6. PMNLs were unstimulated or stimulated by fMLP (A),
E. coli (B), or PMA (C). Representative Western blots and the densitometric evaluation of the blots are shown. Mean values ⫾ SEM. ⴱ, p ⬍ 0.05 vs
unstimulated cells in the absence of resistin.
The Journal of Immunology
FIGURE 9. PMA-induced PMNL oxidative burst. Simplified pathways
showing the kinases involved, the corresponding inhibitors (SB203580:
SB; PD98059: PD; LY294002: LY), and the suggested target of resistin.
MEK1,2: MAPK kinases.
Acknowledgments
We thank Johannes Werzowa and Robert Deicher for providing the
HD-sera.
Disclosures
The authors have no financial conflict of interest.
References
1. Chonchol, M. 2006. Neutrophil dysfunction and infection risk in end-stage renal
disease. Semin. Dial. 19: 291–296.
2. Mahajan, S., O. P. Kalra, K. T. Asit, G. Ahuja, and V. Kalra. 2005. Phagocytic
polymorphonuclear function in patients with progressive uremia and the effect of
acute hemodialysis. Ren. Fail. 27: 357–360.
3. Sardenberg, C., P. Suassuna, M. C. Andreoli, R. Watanabe, M. A. Dalboni,
S. R. Manfredi, O. P. dos Santos, E. G. Kallas, S. A. Draibe, and M. Cendoroglo.
2006. Effects of uraemia and dialysis modality on polymorphonuclear cell apoptosis and function. Nephrol. Dial. Transplant. 21: 160 –165.
4. Vanholder, R., A. Argiles, U. Baurmeister, P. Brunet, W. Clark, G. Cohen,
P. P. De Deyn, R. Deppisch, B. Descamps-Latscha, T. Henle, et al. 2001. Uremic
toxicity: present state of the art. Int. J. Artif. Organs 24: 695–725.
5. Cohen, G., M. Rudnicki, and W. H. Hörl. 2001. Uremic toxins modulate the
spontaneous apoptotic cell death and essential functions of neutrophils. Kidney
Int. 59: S48 –S52.
6. Cohen, G., M. Rudnicki, and W. H. Hörl. 1998. Isolation of modified ubiquitin
as a neutrophil chemotaxis inhibitor from uremic patients. J. Am. Soc. Nephrol.
9: 451– 456.
7. Cohen, G., and W. H. Hörl. 2004. Retinol binding protein isolated from acute
renal failure patients inhibits polymorphonuclear leucocyte functions. Eur.
J. Clin. Invest. 34: 774 –781.
8. Cohen, G., M. Rudnicki, R. Deicher, and W. H. Hörl. 2003. Immunoglobulin
light chains modulate polymorphonuclear leucocyte apoptosis. Eur. J. Clin. Invest. 33: 669 – 676.
9. Cohen, G., M. Rudnicki, F. Walter, T. Niwa, and W. H. Hörl. 2001. Glucosemodified proteins modulate essential functions and apoptosis of polymorphonuclear leukocytes. J. Am. Soc. Nephrol. 12: 1264 –1271.
10. Diez, J. J., P. Iglesias, M. J. Fernandez-Reyes, A. Aguilera, M. A. Bajo,
P. Alvarez-Fidalgo, R. Codoceo, and R. Selgas. 2005. Serum concentrations of
leptin, adiponectin and resistin, and their relationship with cardiovascular disease
in patients with end-stage renal disease. Clin. Endocrinol. 62: 242–249.
11. Otero, M., R. Lago, R. Gomez, F. Lago, J. J. Gomez-Reino, and O. Gualillo.
2006. Leptin: a metabolic hormone that functions like a proinflammatory adipokine. Drug News Perspect. 19: 21–26.
12. Caldefie-Chezet, F., A. Poulin, A. Tridon, B. Sion, and M. P. Vasson. 2001.
Leptin: a potential regulator of polymorphonuclear neutrophil bactericidal action?
J. Leukocyte Biol. 69: 414 – 418.
13. Caldefie-Chezet, F., A. Poulin, and M. P. Vasson. 2003. Leptin regulates functional capacities of polymorphonuclear neutrophils. Free Radic. Res. 37:
809 – 814.
14. Ottonello, L., P. Gnerre, M. Bertolotto, M. Mancini, P. Dapino, R. Russo,
G. Garibotto, T. Barreca, and F. Dallegri. 2004. Leptin as a uremic toxin interferes with neutrophil chemotaxis. J. Am. Soc. Nephrol. 15: 2366 –2372.
15. Montecucco, F., G. Bianchi, P. Gnerre, M. Bertolotto, F. Dallegri, and
L. Ottonello. 2006. Induction of neutrophil chemotaxis by leptin: crucial role for
p38 and Src kinases. Ann. NY Acad. Sci. 1069: 463– 471.
16. Steppan, C. M., S. T. Bailey, S. Bhat, E. J. Brown, R. R. Banerjee, C. M. Wright,
H. R. Patel, R. S. Ahima, and M. A. Lazar. 2001. The hormone resistin links
obesity to diabetes. Nature 409: 307–312.
17. Patel, L., A. C. Buckels, I. J. Kinghorn, P. R. Murdock, J. D. Holbrook,
C. Plumpton, C. H. Macphee, and S. A. Smith. 2003. Resistin is expressed in
human macrophages and directly regulated by PPAR ␥ activators. Biochem. Biophys. Res. Commun. 300: 472– 476.
18. Yang, R. Z., Q. Huang, A. Xu, J. C. McLenithan, J. A. Eisen, A. R. Shuldiner,
S. Alkan, and D. W. Gong. 2003. Comparative studies of resistin expression and
phylogenomics in human and mouse. Biochem. Biophys. Res. Commun. 310:
927–935.
19. Savage, D. B., C. P. Sewter, E. S. Klenk, D. G. Segal, A. Vidal-Puig,
R. V. Considine, and S. O’Rahilly. 2001. Resistin / Fizz3 expression in relation
to obesity and peroxisome proliferator-activated receptor-␥ action in humans.
Diabetes 50: 2199 –2202.
20. Hasegawa, G., M. Ohta, Y. Ichida, H. Obayashi, M. Shigeta, M. Yamasaki,
M. Fukui, T. Yoshikawa, and N. Nakamura. 2005. Increased serum resistin levels
in patients with type 2 diabetes are not linked with markers of insulin resistance
and adiposity. Acta Diabetol. 42: 104 –109.
21. McTernan, P. G., F. M. Fisher, G. Valsamakis, R. Chetty, A. Harte,
C. L. McTernan, P. M. Clark, S. A. Smith, A. H. Barnett, and S. Kumar. 2003.
Resistin and type 2 diabetes: regulation of resistin expression by insulin and
rosiglitazone and the effects of recombinant resistin on lipid and glucose metabolism in human differentiated adipocytes. J. Clin. Endocrinol. Metab. 88:
6098 – 6106.
22. Delamaire, M., D. Maugendre, M. Moreno, M. C. Le Goff, H. Allannic, and
B. Genetet. 1997. Impaired leucocyte functions in diabetic patients. Diabet. Med.
14: 29 –34.
23. Geerlings, S. E., and A. I. Hoepelman. 1999. Immune dysfunction in patients with
diabetes mellitus (DM). FEMS Immunol. Med. Microbiol. 26: 259 –265.
24. Verma, S., S. H. Li, C. H. Wang, P. W. Fedak, R. K. Li, R. D. Weisel, and
D. A. Mickle. 2003. Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction. Circulation 108: 736 –740.
25. Malyszko, J., J. S. Malyszko, P. Kozminski, K. Pawlak, and M. Mysliwiec. 2007.
Elevated resistin is related to inflammation and residual renal function in haemodialysed patients. Nephrology 12: 246 –253.
26. Anderson, P. D., N. N. Mehta, M. L. Wolfe, C. C. Hinkle, L. Pruscino,
L. L. Comiskey, J. Tabita-Martinez, K. F. Sellers, M. R. Rickels, R. S. Ahima,
and M. P. Reilly. 2007. Innate immunity modulates adipokines in humans.
J. Clin. Endocrinol. Metab. 92: 2272–2279.
27. Mojiminiyi, O. A., and N. A. Abdella. 2007. Associations of resistin with inflammation and insulin resistance in patients with type 2 diabetes mellitus. Scand.
J. Clin. Lab. Invest. 67: 215–225.
28. Tokuyama, Y., H. Osawa, T. Ishizuka, H. Onuma, K. Matsui, T. Egashira,
H. Makino, and A. Kanatsuka. 2007. Serum resistin level is associated with
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
experiments assessing the activity of p38 MAPK (Fig. 5), ERK
(p44/42; Fig. 6), and PI3K/Akt (Fig. 7), and found that resistin
abolished the stimulation of PI3K/Akt although it had no effect on
p38- or ERK (p44/42) activation. Therefore, PI3K represents one
possible target of resistin in fMLP-induced PMNL chemotaxis
(Fig. 8).
PD98059 had no significant effect on the stimulation of PMNL
oxidative burst by E. coli in the absence or presence of resistin
(Fig. 4A), suggesting that ERK (p44/42) plays no major role. In
contrast, the inhibitors of p38 MAPK and PI3K significantly attenuated the stimulation by E. coli (Fig. 4A), and resistin had a
strong synergistic effect. Whereas it is widely accepted that the
lack of an additive inhibitory effect points to similar downstream
signaling (52–56), the synergistic effect of resistin might suggest
that its inhibition involves yet to be identified effectors not being
part of the p38- or PI3K-signaling pathways.
The PMA-stimulated oxidative burst was reduced by all inhibitors used (Fig. 4B). The strongest effect was observed when PI3K
was inhibited, pointing to a prominent role of this kinase in the
PMA-stimulated oxidative burst. In line with this finding, Walker
et al. (57) showed that PMA may directly activate PI3K. The activation of p38 MAPK and ERK (p44/42) by PMA was not influenced by resistin, whereas the PI3K/Akt activity was reduced in
the presence of resistin even below basal levels (Fig. 7). These data
suggest that resistin exerts its inhibitory effect on the PMA-stimulated burst by reducing the activity of PI3K (Fig. 9). Of note,
resistin may not directly interfere with PI3K activation, and its
effect may be mediated by unrelated pathways as well. This would
be an explanation for the much stronger attenuation by the direct
inhibitor LY294002 as compared with resistin (Fig. 4B). In conclusion, we show that resistin interferes with the chemotactic
movement and the oxidative burst of PMNLs, and may therefore
contribute to the disturbed immune response in patients with increased resistin serum levels such as uremic and diabetic subjects.
3767
3768
29.
30.
31.
32.
33.
34.
35.
36.
38.
39.
40.
41.
42.
43.
44. Azuma, Y., K. Kosaka, and M. Kashimata. 2007. Phospholipase D-dependent and
-independent p38MAPK activation pathways are required for superoxide production and chemotactic induction, respectively, in rat neutrophils stimulated by
fMLP. Eur. J. Pharmacol. 568: 260 –268.
45. Knall, C., G. S. Worthen, and G. L. Johnson. 1997. Interleukin 8-stimulated
phosphatidylinositol-3-kinase activity regulates the migration of human neutrophils independent of extracellular signal-regulated kinase and p38 mitogen-activated protein kinases. Proc. Natl. Acad. Sci. USA 94: 3052–3057.
46. Zu, Y. L., J. Qi, A. Gilchrist, G. A. Fernandez, D. Vazquez-Abad, D. L. Kreutzer,
C. K. Huang, and R. I. Sha’afi. 1998. p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-␣ or FMLP
stimulation. J. Immunol. 160: 1982–1989.
47. Coxon, P. Y., M. J. Rane, S. Uriarte, D. W. Powell, S. Singh, W. Butt, Q. Chen,
and K. R. McLeish. 2003. MAPK-activated protein kinase-2 participates in p38
MAPK-dependent and ERK-dependent functions in human neutrophils. Cell. Signal. 15: 993–1001.
48. Nagata, T., M. Kansha, K. Irita, and S. Takahashi. 2001. Propofol inhibits FMLPstimulated phosphorylation of p42 mitogen-activated protein kinase and chemotaxis in human neutrophils. Br. J. Anaesth. 86: 853– 858.
49. Chang, L. C., and J. P. Wang. 1999. Examination of the signal transduction
pathways leading to activation of extracellular signal-regulated kinase by formylmethionyl-leucyl-phenylalanine in rat neutrophils. FEBS Lett. 454: 165–168.
50. Camps, M., T. Ruckle, H. Ji, V. Ardissone, F. Rintelen, J. Shaw, C. Ferrandi,
C. Chabert, C. Gillieron, B. Francon, et al. 2005. Blockade of PI3K␥ suppresses
joint inflammation and damage in mouse models of rheumatoid arthritis. Nat.
Med. 11: 936 –943.
51. Heit, B., L. Liu, P. Colarusso, K. D. Puri, and P. Kubes. 2008. PI3K accelerates,
but is not required for, neutrophil chemotaxis to fMLP. J. Cell Sci. 121: 205–214.
52. Fujinaka, Y., D. Sipula, A. Garcia-Ocana, and R. C. Vasavada. 2004. Characterization of mice doubly transgenic for parathyroid hormone-related protein and
murine placental lactogen: a novel role for placental lactogen in pancreatic ␤-cell
survival. Diabetes 53: 3120 –3130.
53. Heuertz, R. M., S. M. Tricomi, U. R. Ezekiel, and R. O. Webster. 1999. C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement. J. Biol. Chem. 274:
17968 –17974.
54. Hsu, M. J., S. S. Lee, S. T. Lee, and W. W. Lin. 2003. Signaling mechanisms of
enhanced neutrophil phagocytosis and chemotaxis by the polysaccharide purified
from Ganoderma lucidum. Br. J. Pharmacol. 139: 289 –298.
55. Krump, E., J. S. Sanghera, S. L. Pelech, W. Furuya, and S. Grinstein. 1997.
Chemotactic peptide N-formyl-met-leu-phe activation of p38 mitogen-activated
protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils. J. Biol. Chem. 272: 937–944.
56. Kuritzkes, D. R., X. Y. Zhang, and E. C. Lin. 1984. Use of phi(glp-lac) in studies
of respiratory regulation of the Escherichia coli anaerobic sn-glycerol-3-phosphate dehydrogenase genes (glpAB). J. Bacteriol. 157: 591–598.
57. Walker, V. G., A. Ammer, Z. Cao, A. C. Clump, B. H. Jiang, L. C. Kelley,
S. A. Weed, H. Zot, and D. C. Flynn. 2007. PI3K activation is required for
PMA-directed activation of cSrc by AFAP-110. Am. J. Physiol. 293:
C119 –C132.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
37.
insulin sensitivity in Japanese patients with type 2 diabetes mellitus. Metabolism
56: 693– 698.
Axelsson, J., A. Bergsten, A. R. Qureshi, O. Heimburger, P. Barany,
F. Lonnqvist, B. Lindholm, L. Nordfors, A. Alvestrand, and P. Stenvinkel. 2006.
Elevated resistin levels in chronic kidney disease are associated with decreased
glomerular filtration rate and inflammation, but not with insulin resistance. Kidney Int. 69: 596 – 604.
Banks, W. A., L. M. Willoughby, D. R. Thomas, and J. E. Morley. 2007. Insulin
resistance syndrome in the elderly: assessment of functional, biochemical, metabolic, and inflammatory status. Diabetes Care 30: 2369 –2373.
Zou, C. C., L. Liang, and F. Hong. 2007. Relationship between insulin resistance
and serum levels of adiponectin and resistin with childhood obesity. Indian Pediatr. 44: 275–279.
Hu, W. L., S. B. Qiao, Q. Hou, and J. S. Yuan. 2007. Plasma resistin is increased
in patients with unstable angina. Chin. Med. J. 120: 871– 875.
Lehrke, M., M. P. Reilly, S. C. Millington, N. Iqbal, D. J. Rader, and M. A. Lazar.
2004. An inflammatory cascade leading to hyperresistinemia in humans. PLoS
Med. 1: e45.
Reilly, M. P., M. Lehrke, M. L. Wolfe, A. Rohatgi, M. A. Lazar, and D. J. Rader.
2005. Resistin is an inflammatory marker of atherosclerosis in humans. Circulation 111: 932–939.
Yannakoulia, M., N. Yiannakouris, S. Bluher, A. L. Matalas, D. Klimis-Zacas,
and C. S. Mantzoros. 2003. Body fat mass and macronutrient intake in relation to
circulating soluble leptin receptor, free leptin index, adiponectin, and resistin
concentrations in healthy humans. J. Clin. Endocrinol. Metab. 88: 1730 –1736.
Nüsken, K. D., J. Kratzsch, V. Wienholz, W. Stöhr, W. Rascher, and J. Dötsch.
2006. Circulating resistin concentrations in children depend on renal function.
Nephrol. Dial. Transplant. 21: 107–112.
Shah, S. V., J. D. Wallin, and S. D. Eilen. 1983. Chemiluminescence and superoxide anion production by leukocytes from diabetic patients. J. Clin. Endocrinol.
Metab. 57: 402– 409.
Inoue, S., Y. Lan, J. Muran, and M. Tsuji. 1996. Reduced hydrogen peroxide
production in neutrophils from patients with diabetes. Diabetes Res. Clin. Pract.
33: 119 –127.
Moon, B., J. J. Kwan, N. Duddy, G. Sweeney, and N. Begum. 2003. Resistin
inhibits glucose uptake in L6 cells independently of changes in insulin signaling
and GLUT4 translocation. Am. J. Physiol. 285: E106 –E115.
Graveleau, C., V. G. Zaha, A. Mohajer, R. R. Banerjee, N. Dudley-Rucker,
C. M. Steppan, M. W. Rajala, P. E. Scherer, R. S. Ahima, M. A. Lazar, and
E. D. Abel. 2005. Mouse and human resistins impair glucose transport in primary
mouse cardiomyocytes, and oligomerization is required for this biological action.
J. Biol. Chem. 280: 31679 –31685.
O’Flaherty, J. T., A. G. Rossi, D. P. Jacobson, and J. F. Redman. 1991. Roles of
Ca2⫹ in human neutrophil responses to receptor agonists. Biochem. J. 277:
705–711.
Steppan, C. M., J. Wang, E. L. Whiteman, M. J. Birnbaum, and M. A. Lazar.
2005. Activation of SOCS-3 by resistin. Mol. Cell. Biol. 25: 1569 –1575.
Coffer, P. J., N. Geijsen, L. M’Rabet, R. C. Schweizer, T. Maikoe,
J. A. Raaijmakers, J. W. Lammers, and L. Koenderman. 1998. Comparison of the
roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function. Biochem. J. 329:
121–130.
RESISTIN INHIBITS PMNL FUNCTIONS