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B activation and cytokine production
Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Correspondence: Michael D. Wheeler, CB# 7365 Mary Elllen Jones Building, Chapel Hill, NC 27599. E-mail: wheelmi{at}med.unc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Vß5
integrin, the co-receptor for adenovirus binding, supporting the
hypothesis that adenovirus transduces Kupffer cells via an Vß5
integrin-dependent mechanism. Indeed, it is shown here that Kupffer
cells express Vß5 integrins. In a functional assay, infection of
isolated Kupffer cells with adenovirus containing superoxide dismutase
or I
B super-repressor blunted LPS-induced nuclear transcription
factor kappa B (NF-B) activation and tumor necrosis factor
(TNF-) production but not IL-10 production. Moreover, superoxide
production was blocked by expression of superoxide dismutase. These
data support the hypothesis that LPS-induced NF-B activation and
TNF- production in Kupffer cells are oxidant-dependent. These
findings suggest that Kupffer cell-targeted approaches may be a
potential therapeutic strategy against many inflammatory diseases
including early alcohol-induced liver injury.
Key Words: alpha V beta 5 integrins • TNF- • superoxide dismutase • IB
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent evidence indicates that blood monocytes/macrophages can be
induced by the growth factor granulocyte-macrophage colony-stimulating
factor (GM-CSF) to express alpha V beta 5 (Vß5) integrin
[5
]. Moreover, the Vß5 integrin acts as a
co-receptor with the coxsackieadenovirus receptor (CAR). The receptor
complex is responsible for adenovirus binding and internalization and
is presumed to be expressed on all adenovirus-permissive cell types
[6
, 7
]. Indeed, adenovirus transduced blood
monocytes/macrophages following treatment with GM-CSF
[8
].
It is hypothesized that several growth factors such as CSFs (i.e.,
GM-CSF and G-CSF) are the cell differentiation switches causing
blood-derived monocytes to become adherent, fixed-tissue macrophages
(e.g., splenic macrophages, alveolar macrophages, and Kupffer cells)
[5
]. In fact, it is well-known that CSFs regulate the
maturation and differentiation of bone macrophage-like osteoclast from
their monocyte precursors [9
]. Moreover, mice deficient
in the CSF-1 gene had reduced numbers of Kupffer cells and dendritic
macrophages markedly, along with a loss of osteoclasts
[10
]. These findings indicate that CSFs are critical
growth factors for producing certain macrophage populations, in
particular, the Kupffer cells, presumably by causing up-regulation of
specific cell-surface adhesion molecules. Thus, it was hypothesized
that Kupffer cells express Vß5, making them permissive to
adenovirus infection.
It is known that activation of Kupffer cells by endotoxin leads to
oxidant production through reduced nicotinamide adenine dinculeotide
phosphate (NADPH) oxidase [11
]. It has been hypothesized
that oxidants generated by Kupffer-cell NADPH oxidase activate nuclear
factor B (NF-B) directly, causing an increase in tumor necrosis
factor (TNF-) production [12
13
14
15
]. This may be
important in the pathogenesis of alcohol-induced liver injury. This
idea is supported by the fact that alcohol-induced injury, as well as
TNF- production in liver, was reduced in rats treated with
Cu/Zn-superoxide dismutase (SOD) delivered via adenovirus
[16
]. Certainly, TNF- is critical for pathogenesis,
because it was shown recently that TNF receptor-deficient mice were
resistant to alcohol-induced liver injury [17
].
However, which cell types are involved remains unclear. Therefore,
recombinant adenoviral vectors containing SOD and IB
super-repressor were used here with isolated Kupffer cells to address
the role of oxidant production in activation of NF-B and production
of TNF-. These data support a clear role of oxidants in the
activation of NF-B and subsequent production of TNF- by Kupffer
cells. Moreover, the data indicate that adenoviral vectors may be
potentially useful, new, therapeutic tools to inhibit Kupffer-cell
activation in disease states.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B super-repressor (Ad.IB) has
been described previously [21
] and was prepared by the
UNC Vector Core. The Ad.IB is a dominant-negative protein that
contains Ser32 
Ala and Ser36 Ala mutations that inhibit
phosphorylation and prevent NF-B dissociation and translocation into
the nucleus. The virus isolates were plaque-purified and propagated in
HEK 293 cells, isolated, concentrated, and titered by plaque assay.
Adenoviral infection
Male Sprague-Dawley rats (250–300 g) were infected with
adenovirus [1x109 plaque-forming units (pfu)] containing
the transgene for HA-tagged Ad.IB or Ad.EGFP. The virus was
diluted in 500 µL 0.9% saline and injected via a tail vein. Kupffer
cells were harvested 3 days after viral infection (see below).
Kupffer cell isolation and culture
Kupffer cells were isolated from naïve Sprague-Dawley
rats (250–300 g) or rats infected with Ad.EGFP (1x109
pfu) 3 days earlier. Briefly, livers were isolated following
pentobarbital anesthesia [60 mg/kg intraperitoneally (i.p.)] and
perfused via the portal vein for 10 min with Krebs-Ringer-HEPES buffer
containing 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4,
25 mM HEPES, 1 mM CaCl2, and 0.016% collagenase (pH 7.4)
followed by 10 min of perfusion with calcium-free buffer containing 0.5
mM EGTA. Liver cells were dispersed by gentle shaking in
phosphate-buffered saline (PBS; pH 7.4, 4°C), and the nonparenchymal
cell fraction was separated from parenchymal cells by centrifugation
through Percoll gradients based on a method developed by Smedsrod and
Pertoft [22
].
Kupffer cells (1x106 cells/ml) were resuspended in Dulbecco’s modified Eagle’s medium (DMEM)-H culture medium containing 10% heat-inactivated fetal bovine serum (FBS), 10 mM HEPES, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate, then seeded onto glass coverslips or 24-well plates, and cultured at 37°C in a 5% CO2 atmosphere. After 1 h, nonadherent cells (e.g., endothelial and stellate cells) were removed by replacement with fresh-culture medium. Phagocytosis of 1 µm latex beads was used to verify that these cells are indeed Kupffer cells. Cells were also incubated with resorufin-ß-D-glucoside to detect contaminating hepatocytes [23 ].
Transgene detection
Immunohistochemical staining
Formalin-fixed, paraffin-embedded sections (6 µm) were mounted
on glass slides. Sections were deparaffinized and rehydrated and then
stained with horseradish peroxidase-conjugated mouse anti-HA-tagged
primary antibody (C12A5; Boehringer Mannhiem, Mannheim, Germany) for 30
min or mouse anti-myeloid cell antigen (ED1; Serotek, Raleigh,
NC) for 1 h. The immunostaining was visualized using the
DAKO immunostaining kit (Dako, Carpinteria, CA). Slides were
counterstained with hematoxylin. Primary antibody dilutions were 1:50
for anti-HA tag antibody and 1:250 for anti-myeloid antigen in PBS with
1% Tween-20.
Biochemical detection of ß-galactosidase
Naïve Kupffer cells were isolated and plated in 24-well
plates and in DMEM-H containing 10% FBS and cultured for 24 h.
Cells were then infected with recombinant adenovirus (0.01–1000
pfu/cell) in the presence of EGTA (2 mM),
Gly-P-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala (cyclical) (Gibco BRL, Life
Technologies, Rockville, MD) peptide (0–2 mg/mL), or P1F6 neutralizing
antibody (5 µg/mL) in DMEM-H containing 2% FBS. After 2 h,
cells were washed with cold PBS and cultured in fresh DMEM-H containing
10% FBS for 24 h. Cells were washed with cold PBS and then
incubated in 250 µL solution containing 5 mM potassium ferricyanide,
5 mM potassium ferrocyanide, 2 mM magnesium chloride, 0.02% Nonidet
P-40 (w/v), 0.01% sodium deoxycholate (w/v), and 1 mg/ml
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal;
Sigma Chemical Co., St. Louis, MO) for 1 h at 37°C. Supernatant
was collected and centrifuged at 10,000 g to remove debris,
and blue color was measured spectrophotometrically at 590 nm.
Measurement of superoxide (O2)
production
Kupffer cell O2 production was
measured by the SOD-inhibitable reduction of ferricytochrome
c [24
]. Cells were plated in 24-well
tissue-culture plates at 106 cells/well and cultured at
37°C for 24 h in DMEM-H with 10% FBS. Cell were then incubated
in the presence of adenovirus (10 pfu/cell) containing
Ad.lacZ, Ad.SOD1, Ad.SOD2, Ad.SOD3, or Ad.IB in DMEM-H
containing 2% FBS. Cells were incubated in the presence of adenovirus
for 2 h and then washed and cultured in fresh DMEM-H containing
10% FBS for 24 h. Supernatant was replaced with Hanks’ balanced
saline solution (HBSS) containing Mg2+ and
Ca2+ supplemented with ferricytochrome
c (0.8 mg/mL, final concentration). Infected Kupffer cells
were then stimulated with lipopolysaccharide (LPS; 10 µg/mL). The
reduction of ferricytochrome c was measured after 30 min in
the presence and absence of purified SOD (85 U/ml). The difference in
absorbance of ferricytochrome c, measured at 550 nm, was
used to calculate the amount of O2
produced, using a molar-extinction coefficient of 18,500.
Electomobility shift assay
Nuclear extracts were isolated as described by Dignam et
al. [25
] with minor modifications. Binding
conditions for NF-B were characterized, and electrophoretic mobility
shift assay (EMSA) was performed as described elsewhere
[26
]. Briefly, nuclear extracts from Kupffer cells (10
µg) were preincubated 10 min on ice with 1 µg poly (dI-dC) and 20
µg bovine serum albumin (BSA; Pharmacia Biotech, Piscataway, NJ) and
2 µl 32P-labeled DNA probe (10,000 cpm/µl;
Cerenkov) containing 1 ng double-stranded oligonucleotide in a
total volume of 20 µl. Mixtures were incubated 20 min on ice and
resolved on 5% polyacrylamide (29:1 cross-linking) and 0.4 x
TBE gels. After electrophoresis, gels were dried and exposed to
X-OMAT LS Kodak film. Data were quantitated by scanning autoradiograms
with GelScan XL (Pharmacia LKB, Uppsala, Sweden).
Measurement of cytokine release in culture media
Isolated Kupffer cells were cultured and infected with
adenoviral vectors as described above. Cells were then incubated with
LPS (1 µg/mL) at 37°C for 4 h for TNF- or 16 h for
interleukin (IL)-10. Standard enzyme-linked immunosorbent assay kits
were used to determine levels of TNF- (Genzyme, Cambridge, MA) and
IL-10 (R&D Systems, Minneapolis, MN) in the culture media.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
super-repressor under the control of the Ad.IB, which was used in
these experiments, because the transgene is fused to an exogenous
peptide marker, making detection convenient using HA antibodies. Three
days after infection, animals were sacrificed, and liver sections were
stained for HA-tagged IB expression. (Fig. 1A
). Nearly 70% of the parenchymal cells expressed HA-tagged
transgene as expected; however, positive staining was also observed in
cells in the hepatic sinusoid (i.e., Kupffer cells, hepatic stellate
cells, and endothelial cells). In serial sections, Kupffer cells were
identified by immunohistochemical analysis using an anti-myeloid,
cell-surface antigen (ED1; Serotek) antibody. Many sinusoidal cells,
which were positive for the HA-tag epitope, were also stained positive
using the ED1 antibody, as indicated by the arrows (Fig. 1A
and 1B)
.
|
To quantify the amount of Kupffer cells transduced by adenovirus, recombinant adenovirus containing the transgene for A. victorius-enhanced Ad.EGFP (1x109 pfu) was injected intravenously. Three days after infection, Kupffer cells were isolated, purified, and plated. Positive cells were counted by fluorescent microscopy and expressed as a percentage of the total number of Kupffer cells isolated. About 15% of the Kupffer cells were transduced under these conditions (unpublished results). Thus, Kupffer cells can indeed be transduced by adenovirus in vivo.
Kupffer cells are transduced in vitro by adenovirus
Because Kupffer cells scavenge and phagocytize particles and
cellular debris, it could be argued that Kupffer cells in
adenovirus-infected animals had taken up cellular debris from infected
hepatocytes, and transgene was detected because it had not yet been
degraded. To test this hypothesis, Kupffer cells were isolated from
naive animals, cultured, and tested for purity using fluorescein
isothiocyanate (FITC)-labeled, 1 µm latex-bead uptake (Fig. 2
). Nearly 100% of the Kupffer cells took up the beads, indicating
that the Kupffer cell cultures were viable and free of contaminating
cells. Next, Kupffer cell cultures were stained with
rusorufin-ß-glucopuranoside, a hepatocyte-specific stain, to test
for contamination by hepatocytes (Fig. 2)
. No viable hepatocytes were
found in Kupffer-cell cultures, indicating that Kupffer cells were
isolated and cultured in the absence of parenchymal cells.
|
|
Vß5 integrinVß5,
neutralizing monoclonal antibody P1F6 (Fig. 3B)
and specific RGD
peptide inhibitors (Fig. 3C)
were used. Isolated Kupffer cells were
infected with Ad.lacZ (10 pfu/cell) in the presence of P1F6
antibody (Chemicon, El Segundo, CA; 5 µg/mL). Transduction of Kupffer
cells was reduced by nearly 80% compared with immunoglobulin G
(IgG)-isotype, antibody-treated control cells. Kupffer cells were also
infected with adenovirus (10 pfu/cell) in the presence of increasing
concentrations of the RGD-peptide inhibitor, cycloSRGDSE (Gibco BRL,
Grand Island, NY), which is a selective inhibitor of Vß5-integrin
binding. Kupffer-cell transduction was inhibited by 
50% with <0.05
mg/ml cycloSRGDSE peptide. Moreover, control peptide
Gly-Arg-Gly-Glu-Ser-Pro (Gibco BRL, Life Technologies) had no effect.
Further, treatment of Kupffer cells with cytochalasin, a potent,
nonspecific inhibitor of phagocytosis/endocytosis, also inhibited
adenoviral transduction (unpublished results). These data are
consistent with the hypothesis that Kupffer cells are transduced by
adenovirus via mechanisms dependent on Vß5 integrins. To rule out
an alternative hypothesis that adenovirus is taken up nonspecifically
by Kupffer cells via phagocytosis, isolated Kupffer cells were
incubated in the presence of PIF6 neutralizing antibody or RGD peptide
inhibitor and given FITC-labeled latex beads (Fig. 4A
). Kupffer cells took up beads in the presence of Vß5
integrin inhibitors, suggesting that inhibition of adenoviral
transduction by these agents was not a result of nonspecific inhibition
of Kupffer-cell phagocytosis. Conversely, phagocytosis of latex beads
was inhibited by pre-treatment with cytochalasin. Collectively, these
data demonstrate that adenoviral transduction of Kupffer cells is
dependent on Vß5 integrins.
|
Vß5 integrins was
tested. Isolated Kupffer cells were immunostained with the F1P6,
antibody-specific for Vß5 integrin, followed by incubation with a
FITC-labeled, anti-mouse antibody (Fig. 4B)
. Indeed, Kupffer cells
stained positively for the expression of Vß5 integrin. Control
experiments included incubation with primary antibody alone and
secondary antibody alone. No immunofluorescence was detected in either
of the control-treatment groups. These data indicate that Kupffer cells
indeed express Vß5 integrins, making the cells permissive to
adenoviral infection and transduction.
Adenovirus encoding SOD and IB inhibit Kupffer-cell oxidant,
NF-B, and TNF- production
To evaluate the therapeutic value of adenoviral transduction of
Kupffer cells and to address the role of oxidants and NF-B in
cytokine production, cells were isolated and infected with
Ad.lacZ (10 pfu/cell) or adenovirus containing the human
genes for cytosolic Ad.SOD1, Ad.SOD2, Ad.SOD3, or Ad.IB, an
inhibitor of NF-B activation. Twenty-four hours after infection,
cells were stimulated with LPS (10 µg/mL), and superoxide was
measured 30 min later (Fig. 5A
). Basal rates of superoxide production were not affected by
adenoviral infection, and LPS increased superoxide production
significantly in uninfected Kupffer cells and cells infected with
Ad.lacZ, as expected. However, superoxide production was
blunted by nearly 60% in Ad.SOD1- and Ad.SOD2-infected cells and 90%
in Ad.SOD3-infected cells. Ad.IB infection had only minimal
effects on LPS-induced superoxide production. Thus, expression of
extracellular SOD attenuated the production of superoxide nearly
completely, whereas IB had little effect.
|
B was measured 4 h after LPS addition by
electromobility shift assay (Fig. 5B)
. NF-B was activated by LPS
nearly fivefold in saline and Ad.lacZ-treated cells. This
phenomenon was blunted nearly 50% in Ad.SOD1-infected cells, whereas
it was completely inhibited by expression of Ad.SOD2, Ad.SOD3, and
Ad.IB.
LPS-induced TNF- production was also evaluated because TNF- is
pivotal in early alcohol-induced liver disease and several other liver
diseases. Ad.SOD1 and Ad.SOD2 infection blunted LPS-induced TNF-
production by >50% compared with saline and
Ad.lacZ-treated cells (Fig. 6A
). Moreover, expression of Ad.SOD3 and IB inhibited TNF-
production completely. It is important that attenuation of TNF-
production correlated positively with inhibition of NFB by SOD and
IB. IL-10 production was also evaluated under these conditions
(Fig. 6B)
. Basal levels of IL-10 (52 pg/mL) were not affected by
adenoviral infection, and LPS caused a significant increase in IL-10
production in saline-treated Kupffer cells as well as
Ad.lacZ-treated cells. Overexpression of SOD or IB had
no effect on the LPS-induced increases in IL-10 production.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Kupffer cells express the adenovirus co-receptor Vß5 integrin
Adenovirus transduction is a receptor-mediated process involving
the CAR, which is nearly ubiquitously expressed, and Vß5
integrins, known as the vitronectin receptors and playing a role in
cell adhesion, thus expressed on many stationary cell types
[34
]. Most likely, this explains why adenovirus
transduces many fixed-tissue cell types readily but transduces
circulating cell types, such as white blood cells, inefficiently
[35
]. Recently, however, it was shown that monocytes,
macrophages, and dendritic cells can be induced to express Vß5 by
GM-CSF and G-CSF, thus making them permissive to adenovirus
transduction [8
, 35
]. Moreover, mice
deficient in genes for CSFs lack mature, resident macrophages,
particularly Kupffer cells and dendritic cells [10
].
From these two independent findings, it was hypothesized that mature
Kupffer cells expressed Vß5 integrins, making them permissive to
adenoviral infection. Indeed, selective inhibitors of Vß5 function
inhibited nearly completely transduction of Kupffer cells by
recombinant adenovirus in vitro (Fig. 3)
. Moreover,
immunofluorescence using antibodies specific for Vß5 demonstrated
clearly the expression of this integrin on naive Kupffer cells (Fig. 4B)
. This is the first study demonstrating that Kupffer cells are
permissive to adenoviral transduction as well as providing a plausible
mechanism for infection. Although many attempts have been made to
infect Kupffer cells with adenovirus, little success has been shown
until now. Positive results were obtained, however, in lung macrophages
and lymphocytes in vitro [35
]. Because
adenovirus transduction requires the expression of alpha V integrins on
the cell surface, it is clear that the isolation procedures and culture
conditions are very important [36
]. The loss of pivotal
receptors in isolation or culture may explain why attempts to transduce
primary cells in culture have not been successful previously.
Kupffer cells can be inactivated by expression of anti-inflammatory
transgenes
Because this work demonstrates that Kupffer cells can be
transduced in culture, the possibility that therapeutic transgenes can
be introduced to inactivate Kupffer cells in particular inflammatory
conditions exists. Recently, it was demonstrated that delivery of
Ad.SOD2 by adenovirus reduced ischemia-reperfusion injury in mouse
liver [37
]. Also, adenovirus containing Ad.SOD1 was used
to reduce early alcohol-induced liver injury and primary nonfunction
following liver transplantation [16
, 38
].
The fact that recombinant adenovirus transduces a significant number of
parenchymal cells is one likely explanation for protection against
oxidative stress in these models. However, many studies have implicated
Kupffer cells in alcohol-induced liver injury and primary nonfunction
[4
, 39
, 40
]. Based on the
observation that Kupffer cells can be transduced in vivo and
the findings that SOD and IB super-repressor blunted activation
of Kupffer cells, conclusions should be re-evaluated to include the
possibility that inactivation of Kupffer cells with recombinant
adenovirus may at least contribute to or be solely responsible for
protection against oxidative stress in the liver by genes delivered via
adenovirus.
Oxidant-NF-B-TNF- axis in Kupffer-cell activation
The finding that Kupffer cells are transduced by adenovirus has
potentially important implications in terms of inflammatory conditions
in vivo but also allows mechanistic questions to be
addressed in vitro. For example, it has been hypothesized
that Kupffer cells are activated by LPS to generate superoxide through
membrane-associated NADPH oxidase [11
, 14
].
Oxidants are also potent activators of NF-B, which drives production
of key inflammatory cytokines such as TNF- [13
,
15
]. The LPS-oxidant-NF-B-TNF- pathway
(Scheme 32) is the proposed mechanism for Kupffer-cell production of TNF-,
which has been shown to be involved critically in several models of
liver injury, including early alcohol-induced liver injury
[17
, 41
]. However, this hypothesis has been
difficult to address because of the lack of specific antioxidants and
inhibitors. Thus, using adenovirus, which contains the transgene for
SOD or IB super-repressor, important mechanistic questions could
be addressed definitively. Here, expression of Ad.SOD3 blunted
LPS-induced superoxide production completely; whereas, intracellular
isoforms of SOD reduced superoxide generation only modestly, and
IB had no effect (Fig. 6)
. It is interesting that all SOD
isoforms and IB blocked LPS- induced NF-B activation nearly
completely and TNF- production (Figs. 7 and 8A). Although the
signaling pathway between LPS stimulation and activation of NF-B is
still largely unknown, recent studies have demonstrated that toll-like
receptors (i.e., TLR-2/4), which associate with the LPS receptor CD14,
can mediate NF-B activation presumbly via IL-1 receptor-associated
kinase (IRAK), NF-B-interacting kinase (NIK), and IB kinases
(IKKs). However, there have been many recent studies demonstrating
several signaling molecules involved in NF-B activation including
IRAK, IKK, and IB to be sensitive to changes in the redox state
of the cell (reviewed by Bowie and O’Neill [42
]).
Moreover, data presented here are consistent with several studies
demonstrating a role of oxidants in NF-B activation and cytokine
production [43
]. Conversely, IL-10 production under
similar conditions was not affected by overexpression of SOD or
IB (Fig. 6B)
. These data suggest that different mechanisms for
TNF- and anti-inflammatory IL-10 production may exist and support
the hypothesis that LPS-induced IL-10 production is independent of
NF-B as shown recently [44
]. These data support
firmly the hypothesis that LPS increases the generation of oxidants
likely via Kupffer-cell NADPH oxidase. Subsequently, oxidants activate
NF-B, which then drives the expression of TNF-. Because NF-B
also plays a critical role in apoptosis in many cell types, it is
reasonable to propose that antioxidant gene delivery may also have
pronounced effects on cell survival. Whether inhibition of NF-B in
Kupffer cells under these conditions plays any role in Kupffer-cell
apoptosis is uncertain.
|
B under the control of Kupffer cell-specific
promoters. Whether this concept will provide therapeutic effects is not
known, but it clearly demonstrates a potential application. It is
important that the potential of developing new Kupffer cell-specific
therapies for many inflammatory conditions, such as alcohol-induced
liver injury, endotoxemia, and primary organ nonfunction, exists.
|
ACKNOWLEDGEMENTS |
|---|
and IL-10. Received April 11, 2000; revised November 14, 2000; accepted November 16, 2000.
|
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vß5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway J. Virol. 69,5951-5958[Abstract]
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 S. W. Luckey, M. Taylor, B. P. Sampey, R. I. Scheinman, and D. R. Petersen 4-Hydroxynonenal Decreases Interleukin-6 Expression and Protein Production in Primary Rat Kupffer Cells by Inhibiting Nuclear Factor-kappa B Activation J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 296 - 303. [Abstract] [Full Text] [PDF]
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 K. M. EDWARDS, M. H. CYNAMON, R. K. R. VOLADRI, C. C. HAGER, M. S. DESTEFANO, K. T. THAM, D. L. LAKEY, M. R. BOCHAN, and D. S. KERNODLE Iron-cofactored Superoxide Dismutase Inhibits Host Responses to Mycobacterium tuberculosis Am. J. Respir. Crit. Care Med., December 15, 2001; 164(12): 2213 - 2219. [Abstract] [Full Text] [PDF]
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