sábado, 20 de marzo de 2010

Kupffer Cells and Their Mediators

Death after multiple trauma occurs either immediately at the scene of
an accident or within hours of the event. Such fatalities are mainly
attributable to the severity of injury or to direct complications from
the primary injury (eg, massive hemorrhage or severe traumatic brain
injury). Patients surviving the initial insult may develop
complications in different organ systems, not necessarily affected by
the primary trauma, within days or even weeks after trauma. In this
group, most patients die because of severe infections and multiple
organ dysfunction syndrome.1
Macrophages play an important role in regulating the immune response
after trauma, shock, and sepsis.2-6 Stimulation of macrophages is
recognized as a physiological reaction of the organism to restore
homeostasis. However, excessive and prolonged activation of
macrophages, in combination with other leukocytes and endothelial
cells, also significantly contributes to the development of the
systemic inflammatory response syndrome and posttraumatic
immunosuppression, which ultimately may result in multiple organ
dysfunction syndrome.7-9 These maladaptive changes in cell function
have been shown to be prevented by female sex steroids (eg,
17ß-estradiol), which are involved in the regulation of macrophage
inflammatory mediator production.10,11
Kupffer cells represent by far the largest population of tissue
macrophages, comprising 80 to 90% of tissue stores. The Kupffer cells
play a key role in the immune response to various low-flow
conditions9,12,13 through secretion of inflammatory mediators that
have significant systemic effects. The proinflammatory cytokine
interleukin-6 (IL-6) for example, which has been shown to participate
significantly in the cascade of events leading to remote organ injury
and increased posttraumatic host susceptibility to sepsis, was shown
to be mainly produced by Kupffer cells after trauma-hemorrhage.9,14
Monocyte chemoattractant protein 1 (MCP-1) is also released by
activated Kupffer cells.15 MCP-1, a member of the CC chemokine family,
plays an important role in the inflammatory response by mediating
directed migration of macrophages and monocytes in several in vivo
inflammatory models (cecal ligation and puncture, peritonitis,
ischemia/reperfusion).15-18 In addition to recruitment of these cells,
MCP-1 can also activate macrophages and endothelial cells.17,19-21
Recent studies have also provided evidence that MCP-1 is significantly
involved in the attraction of neutrophils and the generation of
neutrophil-dependent tissue damage.16,20 Besides Kupffer cells,
several other cell types (lymphocytes, smooth muscle cells, platelets)
are able to produce MCP-1.16,17,19,20,22 Macrophages of other organs
(eg, lung and spleen) are important potential sources of this
chemokine. For example, alveolar macrophages were shown to
significantly increase MCP-1 mRNA expression after an infectious
insult.23
Information on the functions of macrophages, and particularly the
early release of mediators such as MCP-1, should allow for
immunomodulatory intervention aimed at ameliorating the
hyperinflammatory phase, which may lead to the prevention of remote
organ damage and mortality after trauma. We therefore examined whether
Kupffer cells are the main source of systemic MCP-1 after
trauma-hemorrhage and whether the depression of MCP-1 release
contributes to a reduction in remote organ damage. We hypothesized
that Kupffer cells are the main source of MCP-1 production after
trauma-hemorrhage and that modulation of MCP-1 release by E2 or the
estrogen receptor (ER)- agonist propyl pyrazole triol (PPT) will
reduce remote organ damage. To study this, animals were treated with
gadolinium chloride (GdCl3), which is known to ablate Kupffer
cells,9,14 and the effect of Kupffer cell depletion was examined on
systemic MCP-1 as well as on the liver and lung tissue damage after
trauma-hemorrhage. In additional studies, the effect of 17ß-estradiol
(E2) on these parameters and MCP-1 release capacity of different
tissue macrophages (liver, lung, spleen) was determined.

Materials and Methods
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Abstract
Materials and Methods
Results
Discussion
References

Animals and Experimental Groups
All animal studies were performed in accordance with the guidelines of
the National Institutes of Health and were approved by the
Institutional Animal Care and Use Committee, University of Alabama at
Birmingham. Female B57BL/J6 mice (not proestrus), 8 to 12 weeks old
and weighing 19 to 23 g, were obtained from Charles River Laboratories
(Wilmington, MA). These animals were treated with either GdCl3 or
17ß-estradiol (E2). Furthermore, female ER-ß–/– transgenic mice (129
Sve) and corresponding wild-type (WT) animals (12 months, 28 to 32 g
body weight) were included in this study to determine whether ER-
plays a role in the regulation of immune function. These animals were
ovariectomized at 6 weeks of age. The ER-ß KO mice were kindly
provided by Dr. Heather Harris and the Bioresources of Wyeth Research.
These animals received either E2 or the ER- agonist PPT after
trauma-hemorrhage.
Gadolinium Chloride (GdCl3)
Forty-eight hours before trauma-hemorrhage or sham operation, a group
of mice received an intravenous injection of GdCl3 into the tail vein
(10 mg/kg body weight) to ablate Kupffer cells. Control animals
received an intravenous injection of vehicle (0.9% saline) at the same
time point.9,24,25
17-ß Estradiol and ER- Agonist PPT
Subcutaneous administration of the vehicle (dimethyl sulfoxide) was
performed after completion of the sham operation. In trauma-hemorrhage
groups, E2 (50 µg/25 g), PPT (50 µg/25 g), or vehicle (dimethyl
sulfoxide) was injected subcutaneously immediately before onset of
fluid resuscitation.
Trauma-Hemorrhage Procedure
Mice in the trauma-hemorrhage groups were anesthetized with isoflurane
(Minrad, Bethlehem, PA) and restrained in a supine position.26 A
midline laparotomy was performed, which was closed in two layers with
sutures (Ethilon 6/0; Ethicon, Somerville, NJ). Both femoral arteries
and the right femoral vein were cannulated with polyethylene tubing
(Becton-Dickinson, Sparks, MD). Blood pressure was measured via one of
the arterial lines using a blood pressure analyzer (Micro-Med,
Louisville, KY). Within 10 minutes after awakening, animals were bled
through the other arterial catheter to a mean arterial blood pressure
of 35.0 ± 5.0 mm Hg, which was maintained for 90 minutes. At the end
of the procedure, the animals were resuscitated via the venous line
with four times the shed blood volume using Ringer's lactate. After
removing the catheters, the incisions were closed. Sham-operated
animals underwent the same surgical procedures but were neither
hemorrhaged nor resuscitated.
Harvesting Procedures
The animals were again anesthetized with isoflurane and sacrificed 4
hours after sham operation or the completion of resuscitation in the
trauma-hemorrhage groups. Blood was obtained via cardiac puncture
using a syringe coated with ethylenediaminetetraacetic acid (Sigma,
St. Louis, MO). Blood was centrifuged (10,000 rpm, 10 minutes, 4°C)
and the plasma stored at –80°C until further analyzed. Furthermore,
lung, spleen, and liver were removed aseptically.
Determination of Wet-to-Dry Ratios
Wet-to-dry ratios of lung (left lung) and liver (right lobe) were used
as a measure of tissue edema.27 Tissue samples were weighed
immediately after removal (wet weight) and then subjected to
desiccation in an oven at 95°C (Blue M, Asheville, NC) until a stable
dry weight was achieved after 48 hours. The ratio of the wet-to-dry
weight was then calculated.
Myeloperoxidase (MPO) Assay
The accumulation of neutrophils in lung and liver tissue was assessed
by determination of the MPO activity.27 Tissue samples (right lung and
left liver lobe) were collected and frozen in liquid nitrogen and
stored at –80°C until further assayed. For further analysis, frozen
tissue samples were thawed and suspended in 10% phosphate buffer (pH
6.0) containing 1% hexadecyl-trimethylammonium bromide (Sigma). The
samples were sonicated on ice (Sonic Dismembrator; Fisher Scientific,
Hampton, NH). The samples were then centrifuged at 12,000 x g for 15
minutes at 4°C and an aliquot (30 µl) was divided into 180 µl of
phosphate buffer (pH 6.0) containing 0.167 mg/ml o-dianisidine
dihydrochloride and 0.0005% hydrogen peroxide (Sigma). The change in
absorbance at 460 nm was measured spectrophotometrically for 10
minutes. MPO activity was calculated using a standard curve that was
generated using human MPO (Sigma). Protein concentrations of the
samples were determined using a Bradford assay.
Alveolar Macrophages
Lungs were lavaged three times with 1 ml of phosphate-buffered saline
(PBS) containing heparin.28 After centrifugation for 15 minutes at 300
x g at 4°C, cells were resuspended in RPMI 1640 (Life Technologies,
Inc., Grand Island, NY) containing 10% heat-inactivated fetal bovine
serum and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin, and
20 µg/ml gentamicin; all from Life Technologies, Inc.) at a density of
1 x 105 cells/ml. The suspension was then plated in a 96-well plate,
and after 2 hours of incubation (37°C, 95% humidity, and 5% CO2),
nonadherent cells were removed by washing with PBS (Life Technologies,
Inc.). We routinely check the number of adherent cells at the end of 2
hours and have found no significant difference in the number of cells
adhered to plastic surface in 96-well plate in sham and
trauma-hemorrhage animals. Alveolar macrophages in complete RPMI 1640
medium were stimulated with 10 µg of lipopolysaccharide (LPS) (Sigma)
for 24 hours at 37°C, 95% humidity, and 5% CO2. At the end of the
incubation period, the supernatants were removed and stored at –80°C
until analysis was performed.
Isolation of Kupffer Cells
Kupffer cells were isolated as previously described.28,29 In brief,
the portal vein was catheterized with a 27-gauge needle and the liver
was perfused with 20 ml of Hanks' balanced salt solution (HBSS, Life
Technologies, Inc.) at 37°C, which was immediately followed by
perfusion with 15 ml of 0.05% collagenase IV (Sigma) in HBSS with 0.5
mmol/L CaCl2 (Sigma) also at 37°C. The liver was then removed and
transferred to a Petri dish containing the above-mentioned collagenase
IV solution. The liver was minced, incubated for 15 minutes at 37°C,
and passed through a sterile 150-mesh stainless steel screen into a
beaker containing 10 ml of cold HBSS with 10% fetal bovine serum. The
hepatocytes were removed by centrifugation at 50 x g for 3 minutes.
The residual cell suspension was washed twice by centrifugation at 800
x g for 10 minutes at 4°C in HBSS. The cells were resuspended in
Williams' E medium containing 10% fetal bovine serum and antibiotics
(50 U/ml penicillin, 50 µg/ml streptomycin, and 20 µg/ml gentamicin;
all from Life Technologies, Inc.) and layered over 16% metrizamide
(Accurate Chemical, Westbury, NY) in HBSS and centrifuged at 2300 x g
for 45 minutes at 4°C. After removing the nonparenchymal cells from
the interface, the cells were washed twice by centrifugation (800 x g,
10 minutes, 4°C) in Williams' E medium. The cells were then
resuspended in complete Williams' E medium, and plated in a 96-well
plate at a cell density of 5 x 105 cells/ml. After 2 hours of
incubation (37°C, 95% humidity, and 5% CO2), nonadherent cells were
removed by washing with Williams' E medium. We checked the number of
adherent cells at the end of 2 hours and found no significant
difference in the number of adherent cells in sham and
trauma-hemorrhage animals. The cells were then cultured under the
above-mentioned conditions for 24 hours with 10 µg of LPS (Sigma). The
cell-free supernatants were harvested and stored at –80°C until
assayed.
Splenic Macrophages
Splenic macrophages were isolated as previously described.28 Spleens
were gently ground between frosted microscope slides to produce a
single cell suspension. This suspension was centrifuged at 300 x g for
10 minutes at 4°C. The erythrocytes were lysed with lysis buffer and
the remaining cells were washed with PBS by centrifugation (300 x g,
15 minutes, 4°C). After centrifugation, cells were resuspended in RPMI
1640 (Life Technologies, Inc.) containing 10% heat inactivated fetal
bovine serum and antibiotics (50 U/ml penicillin, 50 µg/ml
streptomycin, and 20 µg/ml gentamicin; all from Life Tech-nologies,
Inc.) at a density of 1 x 106 cells/ml. The splenocyte suspension was
used to establish splenic macrophages cultures as described
previously.28 In brief, the splenocyte suspension was plated in a
12-well plate (1 x 106 cells/ml) and after 2 hours of incubation
(37°C, 95% humidity, and 5% CO2), nonadherent cells were removed by
washing with PBS (Life Technologies, Inc.). At the end of 2 hours, the
number of adherent cells was not found to be significantly different
in sham and trauma-hemorrhage animals. Splenic macrophages in complete
RPMI 1640 medium were stimulated with 10 µg of LPS (Sigma) for 24
hours at 37°C, 95% humidity, and 5% CO2. At the end of the incubation
period, the supernatants were removed and stored at –80°C until
analyzed.
Flow Cytometry
IL-6 and MCP-1 concentrations in plasma and cell supernatants were
determined with cytokine bead array inflammatory kits using flow
cytometry according to the manufacturer's instructions (BD Pharmingen,
San Diego, CA). In brief, the adherent cells were cultured for 24
hours, and cell-free supernatants were harvested. Fifty µl of mixed
capture beads were incubated with 50 µl of supernatant and 50 µl of
phycoerythrin (PE) detection reagent for 2 hours at room temperature.
The immunocomplexes were then washed and analyzed using the LSRII flow
cytometer (BD Biosciences, Mountain View, CA). Data processing was
performed using the accompanying FACSDiva and BD CBA software.
Intra-assay specificity was 6.4%, whereas interassay specificity was
3.9%. Calculated values for intra-assay precision and interassay
precision were 8.4 and 6.1%, respectively. The lower detection limits
for IL-6 and MCP-1 were <5 pg/ml.
Treatment of Mice with Anti-MCP-1 Antiserum
Polyclonal anti-mouse MCP-1 antiserum was kindly provided by Dr.
Steven L. Kunkel (University of Michigan, Ann Arbor, MI). To
neutralize MCP-1 activity, 0.5 ml of anti-mouse MCP-1 antiserum or
control serum was injected intraperitoneally 2 hours before
trauma-hemorrhage or sham operation. The time of administration and
the dose of antiserum were selected from previous studies.15,30 It
should also be noted that the biological half-life of the antibody has
been reported to be 36 hours.15
Statistics
Statistical analysis was performed using Sigma-Stat computer software
(SPSS, Chicago, IL). The data were analyzed using one-way analysis of
variance and Tukey's test, and differences were considered significant
at a P value of 0.05. Results are expressed as mean ± SEM of six to
eight animals per group. In studies using knockout animals, three to
four mice were used in each group.

Results
Top
Abstract
Materials and Methods
Results
Discussion
References

GdCl3 or E2 Treatment in Young WT Mice
Plasma MCP-1 and IL-6 Concentrations
Administration of GdCl3 or E2 did not produce any significant change
in plasma MCP-1 (Figure 1A) and IL-6 (Figure 1B) concentrations
compared with vehicle-treated sham mice (P > 0.05). Trauma-hemorrhage
led to a significant increase in plasma MCP-1 and IL-6 levels compared
with the respective sham groups. Pretreatment of animals with GdCl3
before trauma-hemorrhage or administration of E2 after
trauma-hemorrhage resulted in a significant decrease of plasma MCP-1
and IL-6 concentrations
hecho por: Willson A Mendoza C
c.i: 16.959.604
CRF
FUENTE:http://ajp.amjpathol.org/cgi/content/full/169/3/784?ck=nck

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