Cyclosporine A Enhances Leukocyte Binding by Human Intestinal
Microvascular Endothelial Cells through Inhibition of p38 MAPK and
iNOS
PARADOXICAL PROINFLAMMATORY EFFECT ON THE MICROVASCULAR
ENDOTHELIUM*
Parvaneh
Rafiee
,
Christopher P.
Johnson,
Mona S.
Li,
Hitoshi
Ogawa§,
Jan
Heidemann§,
Pamela J.
Fisher§,
Thomas H.
Lamirand§,
Mary F.
Otterson,
Keith T.
Wilson¶, and
David
G.
Binion§
From the Department of Surgery, § Division
of Gastroenterology and Hepatology, Digestive Disease Center and Free
Radical Research Center, Froedtert Memorial Lutheran Hospital,
Milwaukee Veterans Affairs Medical Center, Medical College of
Wisconsin, Milwaukee, Wisconsin 53226 and the ¶ Division of
Gastroenterology, University of Maryland School of Medicine, and
Baltimore Veterans Affairs Medical Center,
Baltimore, Maryland 21201
Received for publication, June 12, 2002
 |
ABSTRACT |
The calcineurin inhibitor cyclosporine
A (CsA) modulates leukocyte cytokine production but may also effect
nonimmune cells, including microvascular endothelial cells, which
regulate the inflammatory process through leukocyte recruitment. We
hypothesized that CsA would promote a proinflammatory phenotype in
human intestinal microvascular endothelial cells (HIMEC), by inhibiting
inducible nitric-oxide synthase (iNOS, NOS2)-derived NO, normally an
important mechanism in limiting endothelial activation and leukocyte
adhesion. Primary cultures of HIMEC were used to assess CsA effects on
endothelial activation, leukocyte interaction, and the expression of
iNOS as well as cell adhesion molecules. CsA significantly increased leukocyte binding to activated HIMEC, but paradoxically decreased endothelial expression of cell adhesion molecules (E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1). In contrast, CsA completely inhibited the
expression of iNOS in tumor necrosis
factor-
/lipopolysaccharide-activated HIMEC. CsA blocked p38 MAPK
phosphorylation in activated HIMEC, a key pathway in iNOS expression,
but failed to inhibit NF
B activation. These studies demonstrate that
CsA exerts a proinflammatory effect on HIMEC by blocking iNOS
expression. CsA exerts a proinflammatory effect on the microvascular
endothelium, and this drug-induced endothelial dysfunction may help
explain its lack of efficacy in the long-term treatment of chronically
active inflammatory bowel disease.
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INTRODUCTION |
The calcineurin inhibitor cyclosporine A (CsA)1 is a
potent immunosuppressive agent that has
formed the pharmacologic cornerstone of solid organ transplantation.
CsA prevents the activation of lymphokine genes essential for T cell
proliferation by disrupting calcium-dependent signal
transduction pathways in leukocytes. Although pharmacologic studies of
CsA have focused primarily on T cell responses, there is emerging
evidence that this agent may exert potent effects on blood vessels,
where it disturbs production of nitric oxide (NO) ultimately promoting
arterial hypertension, inducing long-term vascular dysfunction, and
contributing to obliterative vasculopathy in chronic transplant
rejection (1-4).
Because of the powerful immunosuppressive effect of CsA, and its
success in the prevention of transplant rejection, this agent underwent
extensive clinical evaluation for the treatment of chronic inflammatory
disorders, including inflammatory bowel disease (IBD; Crohn's disease
and ulcerative colitis). Initial studies focused on short courses of
high-dose intravenous CsA therapy, and have demonstrated impressive
success for the treatment of fulminant colitis and the healing of
refractory Crohn's disease fistulas. However, multiple attempts to
convert high-dose CsA to an oral maintenance strategy, akin to the
success in transplant immunosuppression, have paradoxically failed to
demonstrate efficacy (5-11). The precise cellular and molecular
mechanisms that underlie this lack of efficacy in the long term
treatment of chronic intestinal inflammation have remained undefined,
and may result from the emerging profile of adverse effects of CsA on
nonimmune cell populations, including the vascular endothelium.
Endothelial cells lining the microvasculature are now appreciated to
play a central "gatekeeper" role in inflammation through their
ability to recruit circulating immune cells into tissues (12, 13).
Microvascular endothelial activation and adhesion of circulating immune
cells is an early and rate-limiting step in the initiation and
maintenance of the inflammatory response. Advances in vascular biology
have defined an important regulatory role for NO within the
blood vessel during inflammation (14), where endothelial-derived NO
maintains vascular homeostasis through its ability to down-regulate
endothelial cell activation and leukocyte binding (15-19). The effect
of CsA on microvascular endothelial cells, specifically their ability
to regulate leukocyte adhesion during inflammatory activation, is
currently undefined.
The signaling pathways that mediate the inflammatory activation of
nonimmune cells, including endothelial cells, are presently under
intense investigation. The mitogen-activated protein kinase (MAPK)
pathway is a conserved family of eukaryotic signal transduction molecules known to play a major role in the activation of multiple cell
types in response to inflammatory stimuli (20-23). The MAPK superfamily is comprised of the extracellular signal-regulated protein
kinase (ERK1/2; p42/44 MAPK), stress-activated protein kinase (SAPK;
c-Jun NH2-terminal kinase (JNK)), and p38 MAPK. MAPKs are
known to play a key role in the activation of endothelial cells,
leading to an inflammatory phenotype characterized by increased expression of cell adhesion molecules, and enhanced leukocyte binding
(24, 25). Moreover, expression of multiple proinflammatory genes in
endothelial cells is regulated by the transcription factor nuclear
factor B (NFB) (26, 27). The impact of CsA on signal transduction
pathways in nonimmune cell types is distinct from its well
characterized immunosuppressive effect on immune cells (28). However,
the effect of CsA on tissue-specific microvascular endothelial
activation has not been defined.
We characterized the effect of CsA on the activation of human
intestinal microvascular endothelial cells (HIMEC), the microvascular population that mediates leukocyte recruitment during chronic intestinal inflammation in IBD. We hypothesized that CsA would exert a
deleterious, "proinflammatory" effect on HIMEC, through selective
effects on intracellular signaling cascades during inflammatory activation, leading to a loss of NO production and enhanced leukocyte adhesion. To test this hypothesis, we utilized primary cultures of
HIMEC to directly assess the effect of CsA on endothelial-leukocyte interaction. We focused experiments on the effect of CsA on two critical intracellular mechanisms that regulate the inflammatory activation of HIMEC, specifically the expression of iNOS and cell adhesion molecules, as well as the signal transduction pathways that
underlie inflammatory activation of these cells. The results of this
investigation demonstrate that CsA inhibits the production of
iNOS-derived NO in activated HIMEC, normally a key down-regulatory mechanism for limiting endothelial activation induced by tumor necrosis
factor- (TNF-) and lipopolysaccharide (LPS). Furthermore, CsA
differentially inhibited signal transduction pathways during HIMEC
activation, blocking activation of p38 MAPK, a major mechanism for iNOS
gene transcription (29), ultimately resulting in a paradoxical,
proinflammatory effect of this normally powerful immunosuppressive drug.
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MATERIALS AND METHODS |
Patients--
Macroscopically normal mucosal specimens for HIMEC
isolation were obtained from patients undergoing scheduled bowel
resection. The use of discarded human tissues was approved by the
Institutional Review Board of the Medical College of Wisconsin.
Isolation and Culture of Intestinal Microvascular Endothelial
Cells--
HIMEC isolation was adapted from dermal microvascular
endothelium (30, 31). In brief, the surgical specimen was rinsed and
full-thickness samples of intestinal tissue were obtained. Mucosal
strips were dissected, washed, digested in type II collagenase solution
(Worthington) and were then subjected to mechanical compression to
express clusters of microvascular endothelial cells, which were plated
onto fibronectin-coated tissue culture dishes, and grown in HIMEC
medium (MCDB 131 medium (Sigma) supplemented with 20% fetal bovine
serum and endothelial cell growth supplement (Upstate Biochemical, Lake
Placid, NY)). After 7-10 days, microvascular endothelial cell clusters
were physically isolated, and a pure culture was obtained. Endothelial
cultures were verified by modified lipoprotein uptake (Dil-ac-LDL,
Biomedical Technology, Inc., Stoughton, MA) and expression of factor
VIII-associated antigen (32). All HIMEC experiments were carried out
using cells obtained between passages 8 and 14. HIMEC isolates from
resected normal intestinal tissue from six patients were utilized for
this study.
U937 Monocyte-like Leukocytes--
U937 cells, a human
monocyte-like cell line, originally derived from a histiocytic
lymphoma, were obtained from American Type Culture Collection
(Manassas, VA) and maintained in culture with RPMI 1640 medium and 5%
fetal bovine serum. The cells underwent three passages per week and
were used between passages 5 and 10, once re-established in culture.
Pharmacologic Modulation of HIMEC--
CsA (Alexis, San Diego,
CA) was added to the endothelial cultures for specified time periods at
a concentration of 1-10 µM. Additional immunomodulators
were tested including FK506 (1 µM; Calbiochem, San Diego,
CA) and rapamycin (1 µM; Calbiochem). The contribution of
signal transduction pathways to HIMEC activation and gene expression
were defined using specific inhibitors, including: p38 MAPK inhibitor
SB203580 (10 µg/ml; Calbiochem); p42/44 MAPK inhibitor PD98059, which
inhibits MEK (7 µg/ml; Calbiochem); the tyrosine kinase inhibitors
genistein (1 µg/ml; Sigma) and herbimycin (1 µM;
Calbiochem); the antioxidant curcumin (10 µM; Sigma); and the inhibitor of NFB activation Bay 11 (5 µM; Biomol
Research Laboratories, Plymouth Meeting, PA).
Pharmacologic Modulation of Nitric Oxide and Superoxide
Production--
NG-Monomethyl-L-arginine
(L-NMMA, Sigma) (1 mM) or
N-iminoethyl-L-lysine (L-NIL, Alexis
Biochemicals) (1 mM) were added to endothelial monolayers
at the time of induction as competitive inhibitors of nitric-oxide
synthase, respectively. In addition, L-NIL was utilized at
a dosage of 20 µM, which preferentially inhibits iNOS
function (33). Polyethylene glycol-conjugated superoxide dismutase
(PEG-SOD, Sigma) (100 units/ml) was applied to HIMEC monolayers to
preferentially increase degradation or inhibit production of
intracellular superoxide anions during the 24-h activation with
cytokines (TNF-, 100 units/ml; R&D Systems, Minneapolis, MN) and LPS
(1 mg/ml, Escherichia coli 0111:B4, Sigma). PEG-SOD was
applied to the HIMEC monolayers for 2 h prior to and during the
24-h activation period.
Assays of Endothelial Activation Using Leukocyte
Adhesion--
Assays of U937 adhesion to HIMEC were measured as
described previously (31). In brief, endothelial cells were seeded onto fibronectin-coated 24-well tissue culture plates (Corning Glass) at
0.5 × 105 cells/well using HIMEC medium and grown to
confluence over 48-72 h. Unless otherwise noted in the figure legends,
endothelial cells were stimulated with a combination of TNF- (100 units/ml) and LPS (1 µg/ml). After 24 h of activation, HIMEC
monolayers were rinsed and U937 cells (106/ml) were
co-cultured on endothelial monolayers and allowed to adhere at 37 °C
in a 5% CO2 incubator. Following a 1-h incubation, nonadherent cells were removed, and residual cells were rinsed 3 times
with Dulbecco's phosphate-buffered saline (PBS) (Invitrogen) followed by gentle shaking of the tissue culture plate. Monolayers were
fixed and stained using a modified Wright's stain (DiffQuik, Baxter
Scientific, McGraw, IL), and adherent leukocytes were counted in 10 random high power fields (×20) using an ocular grid. Adhesion was
expressed as number of adherent leukocytes/mm2.
In addition to the static adhesion assay described above, an
endothelial-leukocyte low shear stress flow adhesion assay was used to
assess HIMEC activation and function. The endothelial flow chamber was
based on the design of McIntyre and co-workers (34), which allows a
flow of leukocytes at 1 dyne/cm2 over the endothelial
monolayer. Endothelial cells were plated onto fibronectin-coated 35-mm
tissue culture dishes (Corning Glass) at 1 × 105
cells/dish using HIMEC medium and grown to confluence over 48-72 h.
Endothelial cells were analyzed directly or following stimulation with
a combination of TNF- (100 units/ml) and LPS (1 µg/ml). After
24 h, monolayers were rinsed and assayed in a low shear stress
flow chamber (34, 35). U937 cells (1 × 106 per ml of
HIMEC medium) flowing at a rate of 1 dyne/cm2 were
co-cultured over the endothelial monolayers and allowed to adhere at
37 °C, and adhesion was recorded using a CCD camera attached to an
inverted tissue culture microscope for 5 min. Data were analyzed by
counting the number of adherent leukocytes over 10 random high power
fields using a grid and adhesion was expressed as number of adherent
leukocytes/mm2.
Reverse Transcriptase-PCR for iNOS--
iNOS gene
expression was assessed in unstimulated and activated confluent
cultures of HIMEC, with or without CsA and other pharmacologic
inhibitors. Endothelial cells were stimulated with a combination of 100 units/ml TNF
(R&D Systems) and 1 µg/ml LPS (Sigma) for 6 h
at 37 °C. Total RNA was extracted using RNAzol B (Tel-Test,
Friendswood, TX) and quantitated by optical density. 2 µg of RNA was
reverse transcribed using SuperScript II RT (Invitrogen) in a total
reaction volume of 20 µl. 1 µl of reverse transcription product
(cDNA) was PCR amplified with Ampli-Taq DNA polymerase (PerkinElmer Life Sciences) and 0.5 µl each of 10 µM
iNOS forward and reverse primers. In the iNOS reaction, 
-actin
primers were included in the reaction as an internal control for the
efficiency of the reverse transcriptase and the amount of RNA used in
the reverse transcriptase-PCR. Each PCR cycle consisted of a
denaturation step (94 °C, 1 min), an annealing step (60 °C, 1 min), and an elongation step (72 °C, 1.5 min) with a total of 35 cycles, followed by an additional extension step (72 °C, 7 min). The
amplification primers for human iNOS were synthesized based on
published sequences (36, 37). The primer sequences (F = forward,
r = reverse primers) and PCR product sizes were as
follows: iNOS: 5'-TCTTGGTCAAAGCTGTGCTC-3' (F) and
5'-CATTGCCAAACCTACTGGTC-3' (R), 237 bp. PCR products were run on 1%
agarose gels and stained with 0.5 µg/ml ethidium bromide, visualized
under UV light, and photographed.
Measurement of NO Production--
HIMEC (2 × 105) were cultured overnight in 60-mm fibronectin-coated
tissue culture dishes (Corning Glass). After rinsing the monolayers,
medium was replaced with 2 ml of MCDB 131 supplemented with 2% fetal
bovine serum and supernatants were collected after 48 h. Total
NO
/NO
production was assessed by reduction to NO and measurement of chemiluminescence using a Sievers 280 NO analyzer (Sievers Inc., Boulder, CO). Medium, unstimulated cell-conditioned medium, and TNF-
(100 units/ml)/LPS (1 µg/ml) activated cell-conditioned medium were
assessed, and results were recorded as micromolar concentration of NO
production. Results were expressed as the amount of NO production per
milligram of total endothelial cell protein (Bradford assay;
Bio-Rad).
Assessment of Endothelial Reactive Oxygen
Species--
Intracellular generation of reactive oxygen species was
measured using a previously described technique (38). Monolayers of
HIMEC were grown on fibronectin-coated glass coverslips, which had been
precleaned in 70% ethanol and autoclaved. Endothelial cells were
assessed unstimulated or following 24 h stimulation with TNF-
(100 units/ml)/LPS (1 µg/ml), in the presence or absence of CsA
(1-10 µg) 30 min prior to visualization, cells were loaded with 5 mM 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) (Acros Organics/Fisher), a nonpolar compound that freely diffuses into
cells. It is deacetylated by cellular esterases to the membrane impermeable, nonfluorescent derivative
2',7'-dichlorodihydrofluorescein, which in the presence of
intracellular reactive oxygen species and peroxidases, is oxidized
rapidly to the highly fluorescent 2',7'-dichlorofluorescein. Coverslips
of viable HIMEC loaded with DCF-DA were rinsed with medium, inverted,
and visualized on a fluorescence microscope (absorption 504 nm;
emission 529 nm). Photographs of HIMEC DCF-DA fluorescence were taken
using an Olympus camera with a fixed shutter speed of 16 s to
allow for comparison between culture conditions.
Assessment of Cell Adhesion Molecule Surface
Expression--
HIMEC were seeded at 2.5 × 104 cells
per well and grown for 48-72 h in individual fibronectin-coated wells
of a 48-well tissue culture cluster (Corning Glass) until confluence
was reached. Endothelial monolayers were assessed unstimulated, or
following 24 h activation. HIMEC were stimulated with TNF- (100 units/ml)/LPS (1 µg/ml), either alone, or with CsA (1-10 µg/ml) as
indicated under "Results." Saturating amounts of mouse monoclonal
antibodies recognizing human E-selectin, ICAM-1, and VCAM-1 (R&D
Systems) were used to detect HIMEC surface expression of these cell
adhesion molecules. Following a 60-min incubation with anti-cell
adhesion molecule antibodies at 4 °C, monolayers were extensively
rinsed and incubated with goat anti-mouse biotinylated Fab' fragment (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 min
at 4 °C to detect bound primary antibody using whole cell radioimmunoassay. After extensive washing,
125I-streptavidin (80 mCi/ml) was applied to wells to
detect the secondary antibody (39). HIMEC monolayers were lysed with
1.0% Triton X-100 in medium and quantified in a 
-counter. Data from triplicate wells were expressed as the mean of
125I-streptavidin bound (cpm/well). Control experiments
using a nonspecific monoclonal antibody (Mouse IgG1 (MOPC-31c);
Sigma) were performed in parallel at equal concentrations and
incubation conditions.
Electrophoresis and Western Blotting--
Protein concentrations
were determined using a Bradford assay (Bio-Rad). An equal volume of
SDS treatment buffer (125 mM Tris-HCl, pH 6.8, 4% (w/v)
SDS, 20% (v/v) glycerol, 10% (v/v) -mercaptoethanol) was added to
the cell lysate and samples were boiled. Equal amounts of protein were
separated by SDS-PAGE and transferred to nitrocellulose membranes (40).
The membranes were blocked and incubated with specific antibodies
(phosphorylated, nonphosphorylated MAPK (Cell Signaling, New England
BioLabs, Beverly, MA), and iNOS (Santa Cruz Biotechnology, Santa Cruz,
CA)), as specified. Detection was by secondary antibody coupled to
horseradish peroxidase and ECLTM (Amersham Biosciences).
Immunoprecipitation--
Cell homogenate was prepared in lysis
buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM EGTA, 25 mM -glycerophosphate, 1 mM Na3VO4, 25 mM NaF,
10 µg/ml leupeptin, 10 µg/ml aprotinin). Protein A-agarose was used
to immunoprecipitate the cell lysates. Briefly, 50 µl of protein
A-agarose (Santa Cruz) was washed in 1 ml of PBS containing 0.1% (v/v)
Tween 20, resuspended in 100 µl of PBS, and incubated with specific
antibodies for 1-3 h at 4 °C with gentle rotation. Cell lysates
(50-100 µg) were added to protein A-coupled antibody and the complex
was incubated overnight at 4 °C with gentle agitation. After the
immunoprecipitates were recovered by centrifugation, the supernatants
were discarded and the complexes were washed with PBS (3 times) before
further analysis.
Kinase Assay of MAPK Activity--
Assays of kinase activity
were carried out using nonradioactive kinase assay kits (New England
Biolabs) according to the instruction manuals as follows: p42/44 MAP
kinase activity assay: 200 µg of total cellular protein from HIMEC
lysates were incubated with 15 µl of resuspended, immobilized
phospho-p42/44 MAPK (Thr202/Tyr204) monoclonal antibody overnight at
4 °C with gentle agitation. After washing, the pellet was
resuspended in 50 µl of the supplied kinase buffer supplemented with
200 µM ATP and 2 µg of Elk-1 fusion protein for 30 min
at 30 °C. The reaction was then terminated by addition of SDS
treatment buffer and samples were analyzed by SDS-PAGE and Western
blotting using phospho-Elk-1 antibody (Ser383). Western
blots were visualized by ECL.
p38 MAP Kinase Activity Assay--
200 µg of total cellular
protein from HIMEC lysates were incubated with 20 µl of resuspended,
immobilized phospho-p38 MAP kinase monoclonal antibody
(Thr180/Tyr182) overnight at 4 °C with
gentle agitation. After washing, the pellet was incubated with the
supplied kinase buffer with 200 µM ATP and 2 µg
of ATF-2 fusion protein as a substrate for 30 min at 30 °C. ATF-2
phosphorylation was then detected by Western blotting as described
above using phospho-ATF-2 (Thr71) antibody.
Nuclear Protein Extraction and Gel Electromobility Shift
Assay--
Nuclear extracts were prepared as described (41). Briefly,
HIMEC were lysed in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, and 10 ng/µl of each aprotinin and
leupeptin). Cell homogenates were centrifuged (14,000 × g, 1 min, 4 °C) and the pellet was resuspended in buffer
A containing 0.1% (v/v) Nonidet P-40. After centrifugation
(14,000 × g, 10 min, 4 °C), 90 µl of buffer D (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.2 mM
EDTA, 5 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 ng/µl of each
aprotinin and leupeptin) were added to the supernatant (cytosolic
fraction). The pellet was suspended in 60 µl of buffer C (20 mM HEPES, pH 7.9, 25% (v/v) glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol, and 10 ng/µl of each aprotinin and leupeptin).
Samples were incubated on ice for 10 min with frequent vortexing. After
centrifugation (14,000 × g, 10 min, 4 °C), 90 µl
of buffer D was added to the supernatant (nuclear fraction). Protein
concentrations were determined as described above. Nuclear extracts
were incubated with 2 µg/µl poly(dI-dC) and
32P-end-labeled double stranded synthetic
deoxyoligonucleotide probes (for an NFB promotor consensus sequence)
for 30 min at 25 °C in reaction buffer (100 mM EDTA, pH
8.0, 5 M NaCl, 1 M Tris, pH 7.5). The labeled
DNA probes were purified on push columns (Bio-Rad). Protein-DNA
complexes were then resolved in 4% polyacrylamide gels for 2 h at
room temperature in Tris acetate buffer (pH 7.5). Dried gels were
exposed to x-ray film to detect NFB nuclear translocation.
Immunolocalization of p65--
HIMEC were cultured on autoclaved
glass coverslips to confluence. Briefly, unstimulated, CsA-pretreated
(10 µM) HIMEC were activated using TNF-/LPS as above
for 3 h. Cells were fixed with ice-cold methanol, permeabilized
for 5 min with 0.1% (v/v) Triton X-100 in PBS, and blocked with 5%
bovine serum albumin (w/v) in PBS for 1 h. p65 was localized using
anti-p65 antibody followed by incubation with a biotinylated secondary
antibody and fluorescein isothiocyanate-streptavidin (Santa Cruz).
Slides were mounted and visualized using a fluorescence microscope
(Olympus BX-40).
Analysis of Data--
Statistical analysis were performed using
Statview 4.5 and superANOVA software for Macintosh. When single
comparisons were made, t tests were used, applying paired or
unpaired analysis as appropriate. When multiple comparisons between
groups were performed one-way or two-way analysis of variance was used
as appropriate followed by the Student-Newman-Keuls test.
p 
0.05 was considered statistically significant.
| |
RESULTS |
CsA Increases Activated HIMEC-Leukocyte Adhesion--
Primary
cultures of HIMEC interacting with U937 monocyte-like cells were used
to assess the effect of CsA on endothelial activation, and to test the
hypothesis that CsA would cause a proinflammatory effect with enhanced
leukocyte binding in the TNF-/LPS-activated endothelial cells.
Monolayers of HIMEC can be used to assess endothelial function in
vitro, including their ability to undergo activation and binding
of leukocytes through specific interaction of cell adhesion molecules.
U937 cells were used as a target leukocyte population for these assays,
as this established monocyte-like cell line is known to express the
specific cell adhesion molecules VLA-4 and sialyl Lewis X, which
mediate binding to their endothelial ligands VCAM-1 and E-selectin,
expressed on activated HIMEC. HIMEC exhibit a tightly regulated pattern
of U937 monocyte binding that readily increased with overnight
TNF-/LPS activation using an endothelial-leukocyte adhesion assay
(Fig. 1, A and C).
Fig. 1A demonstrates that unstimulated HIMEC bound low
levels of U937, and pretreatment of unstimulated HIMEC with CsA did not
affect leukocyte binding (Fig. 1B). In marked contrast, CsA
pretreatment of the TNF-/LPS-activated HIMEC demonstrated a dramatic
enhancement in U937 binding (Fig. 1D). Quantification of
this enhanced leukocyte binding by CsA-treated HIMEC demonstrated a
significant increase (Fig. 1E). To confirm that this
striking phenomenon of CsA-induced leukocyte hyperadhesion in activated
HIMEC was not an artifact of the static adhesion assay, additional
experiments were conducted in a low shear stress flow adhesion chamber,
which models the physiologic interaction of leukocytes flowing over an
endothelial monolayer. Using this assay, CsA similarly exerted no
effect on the binding of leukocytes to the unstimulated HIMEC
monolayers (Fig. 1F). However, CsA pretreatment of HIMEC
activated for 24 h with TNF-/LPS demonstrated a significant
(p 0.05), 2-fold increase in U937 binding.
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Fig. 1.
A, modified Wright's stain
of unstimulated HIMEC monolayer after 1 h co-culture with U937
monocyte-like cells. Low power (×40), brightfield microscopic view;
firmly adherent U937 nuclei stain dark purple, whereas HIMEC
nuclei stain light purple. HIMEC were seeded at 5 × 105 cells per well and 48 h later were exposed to
106 U937 cells. B, HIMEC monolayer from the
same patient, pretreated with 10 µM CsA for 24 h
prior to co-culture with U937 cells. There is no increase in leukocyte
adhesion to the endothelial monolayer. C, HIMEC
monolayer, stimulated with 100 units/ml TNF- and 1 µg/ml LPS for
24 h prior to co-culture with U937 cells. Ten-fold increase in
leukocyte binding to the HIMEC monolayer following inflammatory
activation is notable. D, HIMEC monolayer pretreated
with CsA prior to stimulation with TNF-/LPS (as above) for 24 h
prior to the 1-h U937 co-culture. Furthermore, a significant increase
in leukocyte binding by HIMEC is noted, above the increase caused by
TNF-/LPS activation demonstrated in panel C. Note that
the increase in leukocyte adhesion following CsA activation only occurs
following activation with TNF-/LPS, and is not seen in the
unstimulated monolayers exposed to this agent. Representative images of
three experiments on HIMEC cultures are shown. E,
static adhesion of U937 monocyte-like cells to HIMEC monolayers in the
absence and presence of CsA. Adhesion assays were performed on
unstimulated monolayers, and endothelial monolayers were pretreated
with CsA prior to stimulation with TNF-/LPS for 24 h prior to
the 1-h U937 co-culture. CsA did not affect binding of the unstimulated
HIMEC, but caused a significant, 2-fold increase in leukocyte binding
in the TNF-/LPS-activated HIMEC. n = 3 total
experiments, each performed with a distinct HIMEC cell line derived
from three different patients, performed in duplicate; an
asterisk denotes a significant difference between cells with
and without CsA (p < 0.05); data are expressed as
mean ± S.E. F, low shear stress flow adhesion of
U937 monocyte-like cells to HIMEC in the absence and presence of CsA.
Adhesion assays were performed on unstimulated monolayers, and
endothelial monolayers preactivated for 24 h with 100 units/ml
TNF- + 1 µg/ml LPS prior to U937 co-culture. The low shear stress
flow adhesion assay measures the binding of leukocytes to the
endothelial surface under physiologic shear conditions of 1 dyne per
cm2. n = 3 total experiments, each
performed with a distinct HIMEC cell line derived from three different
patients, performed in duplicate; an asterisk denotes a
significant difference between the cells treated and not treated with
CsA (p < 0.05); data are expressed as mean ± S.E.
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We then examined the potential mechanisms underlying the
proinflammatory effect of CsA on activated HIMEC and leukocyte
hyperadhesion. Endothelial activation with cytokines and/or LPS results
in increased cell adhesion molecule (CAM) expression, including the
molecules ICAM-1, VCAM-1, and E-selectin. Activation of HIMEC has
previously been demonstrated to result in increased surface expression
of these three adhesion molecules, leading to enhanced interaction with
leukocyte ligands and up-regulation in leukocyte binding to the
endothelium. Previous experiments in our laboratory have demonstrated
that U937 adhesion to HIMEC is dependent on endothelial expression of
E-selectin and VCAM-1. The effect of CsA on CAM expression in
unstimulated and TNF-/LPS-activated HIMEC was assessed using
monoclonal antibodies and a whole cell radioimmunoassay as described
under "Materials and Methods." Fig. 2
demonstrates that resting HIMEC expressed low levels of ICAM-1, and
undetectable levels of E-selectin and VCAM-1. Following activation with
TNF-/LPS, there was a dramatic increase in the cell surface of all
three adhesion molecules. When HIMEC were pretreated with CsA, and then activated with TNF-/LPS, there was a slight, but significant (p 0.05) decrease in the level of expression of all
three CAMs. These data were surprising, given the significant, 2-fold
increase in leukocyte binding, induced by CsA pretreatment in activated HIMEC. These findings suggested that mechanisms other than modulation of CAM surface expression mediate the enhanced leukocyte binding and
proinflammatory effect of CsA on activated HIMEC.
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Fig. 2.
Effect of CsA pretreatment on cell adhesion
molecule surface expression in HIMEC. Activated cultures were
stimulated with 100 units/ml TNF- and 1 µg/ml LPS for 24 h in
the presence and absence of 10 µM CsA. Representative
data from a single experiment is shown; similar results were observed
in three separate experiments. Data are expressed as a mean of
triplicate wells ± S.D. There was a significant difference in the
density of cell adhesion molecule expression in activated HIMEC treated
with and without CsA. Increased cell adhesion molecule expression
correlated with increased leukocyte adhesion in the unstimulated HIMEC
activated by TNF-/LPS. However, the further increase in leukocyte
binding induced by CsA pretreatment did not correlate with an increased
density of the cell adhesion molecules E-selectin, ICAM-1, and VCAM-1,
which demonstrated a significant decrease. An asterisk
denotes a significant difference between the activated cells treated
and not treated with CsA (p < 0.05).
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CsA Inhibits Nitric Oxide Production in Activated HIMEC--
A
major mechanism regulating endothelial activation and homeostasis
involves enhanced nitric oxide production from increased expression of
the inducible nitric-oxide synthase (iNOS, NOS2). We have demonstrated
that iNOS-derived NO down-regulates activation following cytokine
(TNF-, IL-1) and LPS activation of HIMEC (33). To investigate the
effect of CsA on HIMEC NO production and its function, experiments were
performed using static adhesion assays with and without CsA in the
presence and absence of the nonselective NOS inhibitor
L-NMMA. L-NMMA functions as an inactive substrate for the multiple NOS enzyme isoforms, and at 1 mM
concentration prevents NO production from in vitro
endothelial monolayers. When L-NMMA was applied to HIMEC,
there was a significant (p 0.05), greater than
2-fold increase in U937 adhesion, compared with TNF-/LPS-activated HIMEC alone. HIMEC pretreated with CsA then activated with TNF-/LPS demonstrated enhanced leukocyte binding similar to the effect of
nonselective NOS inhibition with L-NMMA. When
L-NMMA and CsA were simultaneously applied to HIMEC prior
to activation, there was no additive effect (33) (Fig.
3A). Likewise,
use of the iNOS-specific inhibitor L-NIL in conjunction
with CsA showed effects similar to L-NMMA, with no further
increase in leukocyte binding (Fig. 3A, right
bars). This suggested that iNOS-derived NO was the major source of
NO production in activated HIMEC.
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Fig. 3.
A, static adhesion of U937
monocyte-like cells to HIMEC pretreated with and without CsA in the
absence and presence of nonspecific and specific iNOS inhibitors. In
the static adhesion assay, 106 U937 monocyte-like cells
were allowed to adhere to HIMEC monolayers over a 1-h time period.
After thorough rinsing, firmly adherent leukocytes are stained using a
modified Wright's stain prior to counting, and data were expressed as
U937 cells per mm2 HIMEC monolayer. Adhesion assays were
performed on unstimulated monolayers, and endothelial monolayers
preactivated for 12 h with 100 units/ml TNF- + 1 µg/ml LPS.
Prior to activation, HIMEC monolayers were pretreated with CsA, or the
nonspecific NOS inhibitor L-NMMA, or the specific iNOS
inhibitor, L-NIL. CsA pretreatment induced a further
significant increase in leukocyte binding by the activated HIMEC. The
CsA induced activation in HIMEC-U937 adhesion was similar to the level
of increased leukocyte binding that was demonstrated by both
nonselective (L-NMMA) and specific iNOS inhibitors (20 µM L-NIL). CsA did not further augment the
effect of the NOS inhibitors, suggesting that it functioned through a
similar mechanism. n = 5 total experiments, each
performed with a distinct HIMEC cell line derived from five different
patients, performed in duplicate; an asterisk denotes a
significant difference between the cells treated and not treated with
CsA (p < 0.05); data are expressed as mean ± S.E. B, effect of CsA on activated HIMEC production of
NO. HIMEC production of NO at baseline and following activation with
100 units/ml TNF- and 1 µg/ml LPS was measured. In addition, HIMEC
monolayer was pretreated with CsA for 24 h prior to activation
with TNF-/LPS. In these experiments, confluent HIMEC monolayers in
60-mm tissue culture dishes were covered with 2 ml of
freshly prepared MCDB 131 medium supplemented with
2% fetal bovine serum. Supernatant was collected after a 48-h time
period and analyzed by chemiluminescence for NO production as described
under "Materials and Methods." NO concentrations were corrected for
background levels in the media and standardized to HIMEC cell protein.
CsA pretreatment of the HIMEC monolayers blocked the increased production of
NO, which normally followed activation with TNF-/LPS.
n = 5 total experiments, each performed on a unique
HIMEC culture. An asterisk denotes a significant difference
between the activated cells treated and not treated with CsA
(p < 0.05); data are expressed as mean ± S.E.
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Because the effect of CsA on activated HIMEC was identical to that of
the NOS inhibitor L-NMMA, which is known to block NO production, we conducted experiments to examine directly the effect of
CsA on NO production by activated HIMEC monolayers. We have previously
established that HIMEC constitutively produce low levels of NO, which
increase 2-3-fold following activation. Detection of very low levels
of NO can be performed using chemiluminescence (33), where the NO
metabolites nitrite, nitrate, and nitrosothiols are measured,
reflecting the original levels of NO production. Pretreatment of HIMEC
monolayers with CsA prior to activation with TNF-/LPS for 48 h
demonstrated a complete inhibition of increased NO production (Fig.
3B). These data strongly suggested that CsA would block
enhanced NO production in activated HIMEC. Because iNOS plays a central
role in the generation of enhanced NO in activated HIMEC, our next
investigation focused on the effect of CsA on HIMEC activation and iNOS
gene expression.
TNF-/LPS Activation of MAPK Family Members in HIMEC--
Our
initial findings demonstrated that CsA exerted a significant effect on
HIMEC during TNF-/LPS-induced activation, but not on the
unstimulated endothelial cells. To further characterize the effect of
CsA on HIMEC activation, we focused our investigation on the MAPK
signaling pathways, which have been implicated in TNF-/LPS induction
of multiple cells types, including endothelial cells. We initially
defined the signaling cascades that mediated TNF-/LPS activation in
HIMEC, using Western blotting of immunoprecipitated cell lysates with
specific phosphoantibodies against all three of the MAPK family members
(Fig. 4, A-C). Using
phospho-specific antibodies for p42/44 MAPK (ERK1/2), p38 MAPK, and
JNK/SAPK, respectively, all three of these signal transduction
molecules revealed rapid phosphorylation in HIMEC following TNF-/LPS
activation. Time course experiments demonstrated that p38 MAPK
underwent a rapid and transient activation, which peaked at 15 min,
whereas p42/44 MAPK activation following TNF-/LPS stimulation peaked
at 30 min (Fig. 4, D and E).
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Fig. 4.
MAPK activation in HIMEC following
stimulation with TNF-/LPS.
Immunoprecipitates of unstimulated and TNF-/LPS-stimulated HIMEC
lysates were analyzed by Western blotting utilizing specific
phosphoantibodies against p42/44 MAPK, p38 MAPK, and JNK.
A, TNF-/LPS-induced phosphorylation of p42/44 MAPK
(ERK1/2); B, phosphorylated p38MAPK was readily
detected following TNF-/LPS activation in HMEC; and
C, similarly, p54 and p46 MAPK (JNK, SAPK) were
phosphorylated by TNF-/LPS in HIMEC. Nonphosphorylated
antibodies were used to detect the total amount of these kinases to
confirm equal loading between lanes. D, a time course
of p38 MAPK activation in HIMEC following TNF-/LPS stimulation
revealed a rapid and transient increase in phosphorylated p38 MAPK that
peaked 15 min following stimulation. E, phosphorylation
of p42/44 MAPK peaked at 30 min following TNF-/LPS stimulation.
Representative images from three separate experiments are shown.
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The Effect of CsA on HIMEC Expression of iNOS--
iNOS expression
following inflammatory stimulation plays an integral role in the
regulation of HIMEC activation. Effect of CsA on the expression of iNOS
mRNA was determined using reverse transcriptase-PCR. TNF-/LPS
activation of HIMEC resulted in a readily detectable signal for the
iNOS gene product (237 bp) and CsA pretreatment inhibited expression of
iNOS (Fig. 5). Experiments were initiated
to evaluate the role of specific MAPK in the expression of iNOS
following TNF-/LPS stimulation. The p38 MAPK inhibitor SB203580
completely inhibited the expression of iNOS mRNA in activated HIMEC, similar to the effect of CsA. The p42/44 MAPK inhibitor PD98059
partially inhibited HIMEC iNOS expression following TNF-/LPS stimulation.
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Fig. 5.
Inhibition of
TNF-/LPS-induced iNOS gene expression in HIMEC
with specific p38 and p42/44 MAPK inhibitors and CsA. Reverse
transcriptase-PCR was used to detect iNOS mRNA expression in HIMEC
following activation with TNF-/LPS for 4 h. Pretreatment of
HIMEC with the specific p38 MAPK inhibitor SB203580, as well as CsA,
completely inhibited the expression of iNOS mRNA in the
TNF-/LPS-activated HIMEC. Pretreatment of HIMEC with the specific
p42/44 MAPK inhibitor PD98059 partially inhibited mRNA expression
for iNOS in these endothelial cells. Activated cultures (+) were
stimulated for 4 h with 100 units/ml TNF- and 1 µg/ml LPS
prior to extraction of RNA. Expression of -actin served as a
semiquantitative reference standard. -Actin was run in the same PCR
reaction as iNOS as an internal control.
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CsA Differentially Inhibits Activation of MAPK Family Members in
HIMEC--
These initial experiments focusing on signal transduction
pathways underlying the expression of iNOS in HIMEC suggested that p38
MAPK was playing the dominant role in TNF-/LPS-mediated expression, and the inhibitory effect of CsA was similar to the specific p38 MAPK
inhibitor SB203580. Western blot analysis of immunoprecipitated cell
lysate demonstrated that the inhibitory effect of CsA on expression of
iNOS was similar to the p38 MAPK inhibitor SB203580, whereas inhibition
of ERK (p42/44 MAPK) with PD98059 failed to inhibit iNOS expression in
TNF-/LPS-activated HIMEC (Fig.
6A). To confirm
that CsA was inhibiting activation of p38 MAPK in
TNF-/LPS-stimulated HIMEC, immobilized phospho-p38 MAPK antibody was
used to immunoprecipitate p38 MAPK in unstimulated and pretreated
TNF-/LPS-stimulated HIMEC. Then in vitro kinase assay was
performed using ATF-2 as a substrate and phospho-ATF-2 antibody
(Thr71) to detect ATF-2 phosphorylation. TNF-/LPS
readily induced p38 MAPK in HIMEC, resulting in subsequent activation
of ATF-2, and this activation was abolished by both CsA and the
specific p38 MAPK inhibitor SB203580 (Fig. 6B). p42/44 MAPK
activation was partially inhibited by CsA (Fig. 6C).
Activation of HIMEC with TNF-/LPS phosphorylated JNK/SAPK, which was
inhibited by genistein, curcumin, and herbimycin. However, CsA exerted
no effect on JNK/SAPK phosphorylation, and this pathway was not further
investigated (Fig. 6D).
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Fig. 6.
A, effect of MAPK inhibitors and
CsA on expression of iNOS protein in TNF-/LPS-activated HIMEC. CsA
and the selective p38 MAPK inhibitor SB203580 significantly decreased
iNOS expression in activated HIMEC. p42/44 MAPK inhibitor PD98059
failed to inhibit TNF-/LPS-induced expression of iNOS protein.
Representative image from three experiments is shown. Mean
densitometric arbitrary units ± S.E. from three HIMEC cultures
derived from separate patients are demonstrated. An asterisk
denotes a significant difference in mean iNOS protein expression
between the TNF-/LPS-activated cells and activated cells pretreated with CsA, SB203580, or PD98059
(p < 0.05). B, effect of CsA on the
TNF-/LPS-induced activation of p38 MAPK in HIMEC. p38 MAPK kinase
activity was assessed by in vitro phosphorylation of the
ATF-2 substrate. TNF-/LPS stimulation of HIMEC phosphorylates and
activates p38 MAPK, which in turn leads to phosphorylation of ATF-2.
CsA pretreatment inhibits this activation similar to the selective p38
MAPK inhibitor SB203580 in HIMEC. A representative image from three
experiments is shown. Mean densitometric arbitrary units ± S.E.
from three HIMEC cultures derived from separate patients are
demonstrated. An asterisk denotes a significant difference
in mean intensity between the TNF-/LPS-activated cells and activated
cells pretreated with CsA or SB203580 (p < 0.05).
C, effect of CsA on the TNF-/LPS-induced activation
of p42/44 MAPK in HIMEC. CsA pretreatment partially inhibits p42/44
MAPK activation in HIMEC, whereas the selective inhibitor PD98059
blocked activation of p42/44 MAPK. These data (A-C) suggest
that p38 MAPK plays a major role in the TNF-/LPS-induced expression
of iNOS in HIMEC, which was significantly blocked by CsA.
Representative images from three experiments are shown. Data represent
results from three separate experiments ± S.E. An
asterisk denotes a significant difference in the cells
treated with either CsA or PD98059 compared with activation alone
(p < 0.05). D, CsA fails to modulate
JNK activation in HIMEC. Unlike its effect on p38 MAPK and p42/44 MAPK,
CsA did not inhibit JNK phosphorylation in the TNF-/LPS-activated
HIMEC. JNK was inhibited by the tyrosine phosphorylation inhibitors
genistein, herbimycin, and curcumin in the TNF-/LPS-activated HIMEC.
Representative images of three separate experiments are shown.
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Effect of CsA on NFB Activation in HIMEC--
NFB plays a
key role in cytokine and LPS activation of endothelial cells (26).
Therefore, we investigated the potential effect of CsA on NFB
activation in HIMEC. TNF-/LPS stimulation readily activated NFB
in HIMEC, as demonstrated by both gel shift analysis (Fig.
7A) as well as nuclear
translocation of the p65 subunit of NFB detected by
immunofluorescence microscopy (not shown). CsA pretreatment of HIMEC
failed to inhibit activation of NFB, unlike Bay 11 and PD98059,
which both blocked NFB activation.
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Fig. 7.
CsA fails to inhibit
TNF-/LPS-induced activation of
NFB in HIMEC. Gel electromobility shift
analysis was performed to determine the effect of CsA on the activation
of HIMEC. NFB inhibitor Bay 11 and PD98059 blocked activation of
NFB, which was not affected by CsA. A representative image of three
experiments is shown.
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CsA Results in Increased HIMEC Oxidant Stress--
Because CsA
significantly increased activated HIMEC-leukocyte binding capacity
independently of cell adhesion molecule surface density, we further
characterized the mechanisms of CsA-induced leukocyte adhesion. Studies
have demonstrated that endothelial activation results in a rapid
increase in the generation of superoxide anions and intracellular
oxidant stress (16, 42). We hypothesized that the loss of iNOS-derived
NO in the CsA-treated HIMEC would similarly lead to enhanced activation
of these cells through an oxidant-mediated mechanism. To assess this
possibility, DCF fluorescence was used to assess the presence of
reactive oxygen species in HIMEC undergoing activation with and without
CsA. DCF-DA is an intravital dye that complexes with intracellular
reactive oxygen species, and can be visualized by fluorescence
excitation. There was low-level oxidant stress detected in unstimulated
HIMEC (Fig. 8A). When HIMEC
were stimulated with TNF-/LPS, oxidant stress was rapidly induced
(data not shown), but after 24 h, levels were only slightly above
baseline (Fig. 8B). However, when HIMEC were treated with
CsA at the time of TNF-/LPS activation, there was a dramatic
increase in reactive oxygen species detected at 24 h (Fig.
8C).
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Fig. 8.
A, high power (×400), fluorescence
microscopic view of live, resting HIMEC monolayers treated with 5 µM DCF-DA for 30 min to detect intracellular reactive
oxygen species. Essentially no fluorescence is detectable.
B, DCF-DA fluorescence for reactive oxygen species in live
HIMEC monolayer (derived from the same patient as Fig. 5A)
stimulated with 100 units/ml TNF- and 1 µg/ml LPS for 24 h. C, DCF-DA fluorescence for reactive oxygen species in
HIMEC monolayer stimulated with TNF-/LPS (as above) pretreated
with CsA prior to activation for 24 h. Increased intracellular
oxyradical stress is demonstrated in the CsA-treated HIMEC following
activation with TNF-/LPS. Representative images of three separate
experiments are shown. Each experiment was performed on a distinct
HIMEC culture.
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Superoxide Scavenger Reverses the Proinflammatory Effect of CsA on
Activated HIMEC--
Because both CsA and NOS inhibition blocked
production of NO and increased intracellular oxyradical stress,
experiments were performed to determine whether this mechanism
contributed to the enhanced HIMEC-leukocyte binding capacity. To
determine whether CsA enhanced leukocyte binding was the result of
increased superoxide radical levels in the TNF-/LPS-activated HIMEC,
we employed a pharmacologic inhibition of superoxide anion with the
scavenger PEG-SOD. In these functional experiments using the
endothelial-leukocyte adhesion assay, concurrent treatment of HIMEC
with PEG-SOD completely reversed the enhanced leukocyte binding induced
by CsA (Fig. 9). These data imply that
excess superoxide generated in the activated endothelium treated with
CsA plays a critical role in the enhanced leukocyte binding.
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Fig. 9.
Reversal of CsA-induced hyperadhesion of U937
monocyte-like cells to HIMEC pretreated with the superoxide scavenger
PEG-SOD. Adhesion assays were performed on endothelial monolayers
preactivated for 24 h with 100 units/ml TNF- + 1 µg/ml LPS
prior to U937 co-culture. PEG-SOD was applied concurrently during the
24-h activation period. An asterisk denotes a significant
difference between cells treated with and without CsA
(p < 0.05); n = 3 total experiments,
performed in duplicate; data are expressed as mean ± S.E.
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Effect of FK506 and Rapamycin on HIMEC-Leukocyte
Interaction--
Additional calcineurin inhibitors and pharmacologic
agents have emerged in transplant immunosuppression with the potent
ability to block rejection, and have undergone preliminary testing in trials of patients with chronic inflammation and IBD. FK506 is known to
inhibit calcineurin, Na+K+-ATPase,
FK506-binding protein activity, and proliferation of lymphocytes.
Rapamycin is a macrolide antibiotic with potent antifungal and
immunosuppressive qualities. It forms a stable complex with the
FK-binding protein (FKBP-12) with a high affinity to the mammalian target of rapamycin. This interaction causes dephosphorylation and
inhibition of p70S6 kinase, ultimately inhibiting cell cycle re-entry
and expansion of lymphocyte populations, leading to its immunosuppressive effect in transplantation. Both FK506 and rapamycin have been implicated in causing endothelial cell dysfunction, similar
to the effect of CsA (43). We performed experiments to determine
whether the proinflammatory effect of CsA on the microvascular
endothelium was unique, or would be seen in the other most frequently
used potent transplantation immunosuppressive compounds. Flow
leukocyte-adhesion assays were performed on HIMEC pretreated with CsA,
FK506, and rapamycin. Both FK506 and rapamycin induced a pattern of
enhanced U937 adhesion, which was similar to CsA in HIMEC activated
with TNF-/LPS (Fig. 10). This
preliminary investigation suggests that endothelial dysfunction and
enhanced inflammatory activation is a common feature shared by all
three of these major immunosuppressive compounds. The mechanisms
underlying the enhanced leukocyte binding in the FK506 and
rapamycin-treated endothelial cells were not specifically defined.
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Fig. 10.
Low shear stress flow adhesion of U937
monocyte-like cells to HIMEC in the absence and presence of the
immunosuppressive agents CsA, FK506, and rapamycin. Adhesion
assays were performed on endothelial monolayers preactivated for
24 h with 100 units/ml TNF- + 1 µg/ml LPS prior to U937
co-culture. All three of these immunosuppressive agents exerted a
proinflammatory effect on the HIMEC monolayers, and caused similar
rates of increased U937 leukocyte adhesion. The low shear stress flow
adhesion assay measures the binding of leukocytes to the endothelial
surface under physiologic shear conditions of 1 dyne/cm2.
n = 3 total experiments, each performed with a distinct
HIMEC culture derived from three different patients, performed in
duplicate; an asterisk denotes a significant difference
between cells treated with and without the specifically designated
immunosuppressive agent (p < 0.05); data are expressed
as mean ± S.E.
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DISCUSSION |
We have demonstrated that the immunosuppressive agent CsA affects
gene expression patterns in human intestinal microvascular endothelial
cells following inflammatory activation. The CsA effect was mediated
through differential inhibition of signaling pathways, ultimately
resulting in a paradoxical proinflammatory phenotype characterized by
enhanced leukocyte binding. As expected, stimulation of HIMEC with
TNF- and bacterial lipopolysaccharide resulted in activation of the
MAPK cascades and NFB, and enhanced expression of the
proinflammatory cell adhesion molecules ICAM-1, VCAM-1, and E-selectin.
Activated HIMEC demonstrated an established enhanced adhesive
interaction with leukocytes, which represents an important early
control step in the inflammatory response. In addition to the increased
expression of these proinflammatory cell adhesion molecules, activation
with TNF-/LPS also induced the expression of iNOS and increased NO
production, a key mechanism that ultimately mediates the
down-regulation of HIMEC activation. Although treatment of HIMEC with
CsA caused a modest decrease in the expression of proinflammatory
molecules (CAM), it exerted a potent, and complete inhibition of the
anti-inflammatory enzyme iNOS in these microvascular endothelial cells.
The net effect of CsA on HIMEC activation was a loss of NO production
and a significant, proinflammatory increase in leukocyte binding, an
unexpected, paradoxical effect for this drug, which is normally used
clinically to inhibit inflammatory responses. Although CsA has been
previously demonstrated to exert an inhibitory effect on the expression
of iNOS in other cell types, including vascular smooth muscle cells and
mesangial cells, this is the first report of its direct effect on the
microvascular endothelium, a nonimmune cell population that plays a
critical regulatory role in inflammatory responses. The specific
inhibitory effect of CsA on iNOS expression in nonimmune cells has not
been completely defined, which prompted our investigation of the
intracellular signaling mechanisms involved in HIMEC activation and the
effect of CsA on these activation pathways.
Perhaps the most important aspect of this investigation centers on the
characterization of differential inhibition of MAPK signaling pathways
by CsA. CsA selectively inhibited p38 MAPK during the activation of
intestinal microvascular endothelial cells, which ultimately promoted a
proinflammatory phenotype in these cells. An equally important aspect
of this investigation was the characterization of both proinflammatory
and anti-inflammatory gene expression profiles simultaneously induced
by cytokine and LPS activation of microvascular endothelial cells.
TNF- and LPS readily activated MAPK family members as well as NFB
in HIMEC. MAPK and NFB activation resulted in proinflammatory
expression of CAM, and a simultaneous expression of iNOS, a key
regulatory mechanism for limiting activation in HIMEC. Although CsA
affected multiple gene expression patterns, it exerted its most
powerful effect on the expression of the regulatory mechanism
(i.e. iNOS expression) through a potent inhibition of p38
MAPK activation.
CsA is highly effective as an immunomodulator, but has demonstrated
mixed results in the treatment of long term chronic inflammation, particularly inflammatory bowel disease. Much of the investigation regarding the therapeutic efficacy of CsA has focused on the effect of
this compound on lymphocytes, specifically T cells and the generation
of cytokines, with most research focusing on interleukin 2 (28). Our
investigation of the pharmacologic activity of CsA on nonimmune cell
populations suggests that this compound also exerts potent effects on
microvascular endothelial populations, which play key regulatory roles
in the inflammatory process. Our work suggests that CsA exerts multiple
effects on the blood vessel wall, the net result of which appears to be
an enhancement of inflammation through increased leukocyte binding activity.
Selective inhibition of signal transduction pathways is a rapidly
emerging strategy in molecular medicine. Design of compounds, which
will effect specific cellular activation pathways underlying disease
mechanism, while limiting adverse reactions, is the ultimate goal of
drug design. Inhibition of JNK and p38 MAPK has already been
demonstrated to show clinical efficacy in short term trials of severely
ill patients with Crohn's disease (44). Likewise, the short term use
of intravenous CsA has shown efficacy in the treatment of patients with
both ulcerative colitis and Crohn's disease (7-8), whereas long term
use has failed to demonstrate clinical improvement.
Although experiments examining the role of the vasculature in animal
models of intestinal inflammation are essential, there are important
distinctions in the nitric oxide biology of rodents, which may not
allow for a direct extrapolation of findings regarding our
understanding of human microvascular function during intestinal inflammation (45). Experiments in human intestinal inflammation must
rely on in vitro approaches, and the use of tissue-specific microvascular endothelial cell cultures represents an essential strategy for gaining insight into molecular and cellular mechanisms affected by pharmacologic strategies. Our investigation of HIMEC activation and the proinflammatory effect of CsA may offer a potential mechanism for the lack of efficacy of long term CsA therapy in IBD. We
have demonstrated previously the lack of iNOS-derived NO in endothelial
cells isolated from chronically damaged areas of IBD bowel in contrast
to adjacent normal areas of intestine. This loss of iNOS-derived NO
impairs the ability of microvascular endothelial cells to down-regulate
inflammatory activation, and may thus contribute to chronic intestinal
inflammation. iNOS-derived NO is preserved in uninvolved areas of gut
in IBD patients, suggesting that an intact ability to produce NO in the
microvasculature prevents worsening or extension of the disease process
(46). Our present investigation suggests that CsA adversely affects NO
production from the intestinal microvasculature, impairing vascular
homeostasis, leading to enhanced leukocyte recruitment, and worsening
uncontrolled, chronic intestinal inflammation. The effect of CsA on
HIMEC activation may also offer mechanistic insights into the high
prevalence of IBD in solid organ transplantation, where transplant
recipients treated with long term calcineurin inhibitor
immunosuppression (i.e. CsA and FK506) experience rates of
clinical IBD 10 times greater than in the general population (47).
Defining the beneficial and deleterious effects of established
pharmacologic agents represents an important goal in improving therapeutic approaches. Our present investigation may provide a
mechanistic explanation regarding a proinflammatory effect of CsA on
the organ-specific microvascular endothelium, and the failure of this
compound in the long term treatment of chronic inflammatory bowel
disease. If CsA blocks the generation of iNOS-derived NO in the
activated intestinal microvasculature, then replacing endogenously produced NO with pharmacologic NO delivery may offer a long term strategy for improved immunomodulatory therapy using calcineurin inhibitors. In addition, we have shown that iNOS-derived NO functions as an antioxidant to quench superoxide anions generated during HIMEC
activation. Endothelial delivery of antioxidant, in addition to
coupling CsA to a pharmacologic NO donor compound, may restore the
function of NO whose production was inhibited by CsA. Conjugation of NO
donor compounds with pharmacologic agents has been initiated, as
NO-mesalamine has already undergone preclinical investigation, and has
demonstrated improved efficacy and safety in the treatment of animal
models of IBD (48).
| |
ACKNOWLEDGEMENTS |
We thank Dr. Bellur Seetharam, Medical
College of Wisconsin, for critical review of the manuscript and Cara
Olds for technical assistance with the gel electromobility shift experiments.
| |
FOOTNOTES |
*
This work was supported in part by National
Institutes of Health Grants DK02417, DK 56234 (to D. G. B.),
DK02469 (to K. T. W.), and the Crohn's and Colitis
Foundation of America (to D. G. B.). Presented in abstract form at
the American Gastroenterological Association and Digestive Disease
Week, San Diego, CA, May 21-24, 2000.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.
To whom correspondence should be addressed: Medical College of
Wisconsin, Division of Gastroenterology and Hepatology, Froedtert Memorial Lutheran Hospital, 9200 West Wisconsin Ave., Milwaukee, WI
53226. Tel.: 414-456-6845; Fax: 414-456-6214; E-mail:
dbinion@mcw.edu.
Published, JBC Papers in Press, July 10, 2002, DOI 10.1074/jbc.M205826200
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ABBREVIATIONS |
The abbreviations used are:
CsA, cyclosporine A;
HIMEC, human intestinal microvascular endothelial
cells;
NO, nitric oxide;
NOS, nitric-oxide synthase;
VCAM-1, vascular
cell adhesion molecule-1;
ICAM-1, intercellular adhesion molecule-1;
MAPK, mitogen-activated protein kinases;
SAPK, stress-activated protein
kinase;
JNK, c-Jun NH2-terminal kinase;
ERK, extracellular
signal-regulated kinase;
TNF-, tumor necrosis factor ;
LPS, lipopolysaccharide;
NFB, nuclear factor B;
IBD, inflammatory
bowel disease;
DCF-DA, dichlorodihydrofluorescein diacetate;
L-NMMA, NG-monomethyl-L-arginine;
L-NIL, N-iminoethyl-L-lysine;
PEG-SOD, polyethylene glycol-conjugated superoxide dismutase;
ROS, reactive oxygen species;
PBS, phosphate-buffered saline;
CAM, cell
adhesion molecule;
iNOS, inducible nitric-o
TOP
ABSTRACT
INTRODUCTION
