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Inhibits Activation-Induced Expression of E- and P-Selectin on Endothelial Cells
Protein Design Labs, Mountain View, CA 94043
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, and PMA. Induction of E-selectin is blocked by pretreatment of
endothelial cells with IL-4 or TGF-ß, both of which have
antiinflammatory properties in vivo. In addition to its well-known
proinflammatory activities, IFN-
also has antiinflammatory effects
in vivo, one of which is inhibition of neutrophil recruitment. To
determine whether IFN- inhibits neutrophil recruitment by inhibiting
adhesion molecule expression, the effect of IFN- on
activation-induced cell adhesion molecule expression by cultured HUVEC
was evaluated. Pretreatment of endothelial cells with IFN- for 24 to
72 h before 6- to 24-h activation with IL-1ß, TNF-, or PMA
resulted in significantly reduced levels of cell surface E-selectin,
although levels of ICAM-1 and VCAM-1 were the same or increased. The
reduction of cell surface E-selectin levels under these conditions was
reflected in reduced levels of E-selectin mRNA, indicating an effect at
the transcription level or RNA stability. Interestingly, the increase
of cell surface P-selectin expression due to IL-4 treatment of HUVEC
was also inhibited by IFN-, while constitutive levels of P-selectin
were not. These results suggest that the inhibition of neutrophil
recruitment by IFN- in vivo may be due, in part, to the ability of
IFN- to inhibit E- and P-selectin up-regulation. Furthermore, these
findings emphasize the process of leukocyte recruitment as an important
step through which IFN- can direct the character of inflammatory
reactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are well known and include a
central role in macrophage activation, induction, and enhancement of
MHC class I and II Ags on a wide variety of cell types, differentiation
of B cells, and regulation of the proliferation and function of
activated T cells (reviewed in 1 . IFN- production is a
characteristic of Th1 cells, Th cells that drive delayed-type
hypersensitivity reactions. Th1 responses are required for protection
from intracellular pathogens and are most likely important for the
development of certain autoimmune syndromes (2).
Despite understanding many of the proinflammatory activities of
IFN-, the role of IFN- in autoimmune and inflammatory diseases
has been difficult to elucidate. Conflicting data have been observed in
many in vivo models. For example, IFN- treatment protects C57BL/6J
mice from experimental allergic encephalomyelitis, which some describe
as a model of multiple sclerosis; however, treatment of patients with
IFN- has led to an exacerbation of the disease (3, 4). While Ab to
IFN- delays the lupus-like nephritis in (NZB x
NZW)F1 mice, it has no effect on the nephritis in the
MRL/lpr-lpr mouse model of lupus (5, 6). Abs to IFN- also
have disparate effects in the adjuvant arthritis model in rats,
depending on when therapy is given (7, 8). Together these data have
suggested that IFN- may have both anti- and proinflammatory
activities, the interactions of which are not well understood (1, 4).
The ability of IFN- to modulate leukocyte recruitment is one
function for which both anti- and proinflammatory activities have
been ascribed. Although IFN- is a potent inducer of lymphocyte and
monocyte recruitment (9, 10), several reports have suggested that
IFN- may also block neutrophil recruitment (11, 12, 13). Several years
ago, Hallmann, in collaboration with Jutila, Amento, and
Butcher, observed that treatment of cultured endothelial cells with
IFN- resulted in reduced neutrophil adherence to IL-1ß-activated
endothelial cells (14). To determine the mechanism by which IFN-
inhibits neutrophil-endothelial cell adhesion, we tested the ability of
IFN- to modulate the expression of endothelial cell adhesion
molecules for neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant IFN-, TNF-, IL-1ß, and IL-4 were
obtained from R&D Systems (Minneapolis, MN) and reconstituted, as
suggested by the manufacturer, except for IFN-, which was
reconstituted in 0.1% BSA in PBS. PMA was obtained from Clonetics (San
Diego, CA). Human thrombin and mouse IgG1 and IgG2b myeloma proteins
MOPC 21 and MOPC 141 were obtained from Sigma (St. Louis, MO). mAb
E-1E4, anti-human E-selectin (murine IgG1), has been previously
described (15). E-1E4 was biotinylated by labeling with long chain
biotin, according to the method described by Pierce (Rockford,
IL). AF-2 (murine IgG2b), a neutralizing mAb to human IFN-, was
kindly provided by Nick Landolfi (Protein Design Labs, Mountain View,
CA). Anti-human VCAM-1 was obtained from PharMingen (San Diego, CA);
anti-ICAM-1 from Coulter (Miami, FL); and human P-selectin, WAPS
12.2, from Endogen (Woburn, MA). J13C2.4 is a mouse IgG1
anti-HLA-DR Ab, generated in our laboratory, and purified from
serum-free supernatants, as described (15).
Endothelial cell cultures
HUVEC were obtained from Clonetics. These cells were cultured in endothelial cell growth media (EGM;2 Clonetics), containing bovine brain extract (12 µg/ml), human epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), gentimicin (50 µg/ml), amphotericin B (50 ng/ml), and 2% FBS in MCDB-131 medium, for one to eight passages, and subcultured with trypsin-EDTA, as described by the manufacturer. Experiments were performed by culturing HUVEC in 96-well plates (Falcon; Becton Dickinson, Lincoln Park, NJ), in the presence of various cytokines or activators for the indicated times.
Adhesion assays
HL-60 cell binding to HUVEC was performed essentially as
described (15, 16). Briefly, HUVEC were cultured in 96-well plates in
EGM with or without IFN- for 24 h, then with or without PMA for
an addtional 4 h, washed, and resuspended in 50 µl/well assay
buffer (10% adult bovine serum/10% normal rabbit serum/10 mM HEPES,
pH 7.2/RPMI). Fluorescently labeled HL-60 cells were prepared as
previously described (15) and resuspended in assay buffer containing
0.25 µg/ml anti-CD18 Ab, NA8, at 2 x 106
cells/ml. Assays were initiated by the addition of 50 µl HL-60 cells
to HUVEC for a final volume of 0.1 ml, while plates were rotated at 40
rpm (Innova 200; New Brunswick, Edison, NJ). After 15 min at room
temperature, unbound HL-60 cells were removed by washing plates four
times with 0.2 ml RPMI/well. Bound cells were fixed by the addition of
0.1 ml 1% paraformaldehyde (Sigma) in PBS. Plates were analyzed with a
Microplate Fluorometer (model 7620; Cambridge Technology, Watertown,
MA), and the relative binding of HL-60 cells was determined by
measuring the amount of fluorescence at 530 nm, using an excitation of
485 nm.
Cell-based ELISAs
Microtiter plates containing treated HUVEC were washed with 200 µl/well PBS and inverted until plates were dry. Wells were blocked by incubating with 200 µl 1% Blotto (Pierce) in PBS for 30 min. Primary Abs, including anti-E-selectin, anti-VCAM-1, anti-ICAM-1, anti-HLA-DR, anti-P-selectin, isotype control Abs, or biotinylated anti-E-selectin, were then added to washed plates at 0.5 to 2 µg/ml in 0.05% Blotto/PBS for 2 h. Plates were again washed in 0.05% Blotto/PBS, and 50 µl peroxidase-conjugated anti-mouse IgG (Promega, Madison, WI), diluted 1/3000, or peroxidase-conjugated streptavidin (Pierce), diluted 1/1000, in 0.05% Blotto/PBS, was applied for 1 h. After washing, 100 µl TMB substrate (Kirkegaard and Perry, Gaithersburg, MD) was added and color was developed for 5 to 10 min. A total of 100 µl 1 M H2SO4 was then added and absorbance was read at 450 nm (subtracting the background absorbance at 600 nm) with a Dynatech (Chantilly, VA) plate reader.
Flow cytometry
HUVEC were released from tissue culture plates by brief incubation in trypsin-EDTA (Clonetics). After washing cells in EGM, flow-cytometry experiments were performed by incubation of 5 x 105 cells in 0.1 ml FACS buffer (0.1% BSA, 10 mM NaN3 in PBS) with 10 to 100 ng primary Ab for 1 h at 4°C. After washing, 50 µl of goat F(ab')2 anti-mouse IgG-phycoerythrin conjugated (Biosource, Camarillo, CA), diluted 1/500 in FACS buffer, was added and incubated for 30 min before washing and fixation in 1% paraformaldehyde. Cells were analyzed with a FACScan (Becton Dickinson, San Jose, CA), according to standard procedures.
Quantitative RT-PCR
The procedure for utilizing known concentrations of competitive
DNA fragments in PCR reactions for precisely measuring mRNA levels (as
described in Refs. 17–19) was obtained from Clontech Laboratories
(Palo Alto, CA). HUVEC were cultured in 24-well plates, as described
above, in the presence of IFN- (0 or 250 ng/ml), for 48 h, and
PMA (100 ng/ml) for an additional 4 h. Cells were solubilized by
direct treatment with TRIzol reagent (Life Technologies, Gaithersburg,
MD), and total RNA was prepared according to the manufacturer’s
instructions. The first-strand cDNA synthesis was performed with
SuperScript II RNase H- reverse transcriptase (Life
Technologies) using random hexadeoxynucleotides (Pharmacia, Piscataway,
NJ) as primer. PCR reactions contained a fixed amount of cDNA, various
known concentrations of E-selectin, ICAM-1, or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) competitor DNAs, and
appropriate primers. The reaction was performed with TaqPlus polymerase
(Stratagene, La Jolla, CA) at 94°C for 5 s, 57°C for 5 s,
and 72°C for 1 min with 30 cycles. Competitor DNA and PCR primers for
GAPDH were obtained from Clontech, and result in native and mimic PCR
products of 983 and 630 bp, respectively. The PCR primers for
E-selectin were 5'-ACCTCCACGGAAGCTATGAC-3' and
5'-TCCCAGATGAGGTACACTGA-3', which produce a 796-bp native PCR
product; and those for ICAM-1 were 5'-ACATGCAGCACCTGTGA-3' and
5'-AGAAGGAGTCGTTGCCATAG-3', which produce a 673-bp native PCR
product. Competitor DNAs for E-selectin and ICAM-1 were constructed by
deleting 240- and 213-bp fragments, respectively, within the regions
amplified by the above PCR primers from the corresponding cDNAs to
yield mimic PCR fragments of 556 and 460 bp, respectively. The PCR
products were separated on a 3% agarose gel (Life Technologies), and
the intensity of native and mimic PCR products for each reaction (each
containing a different amount of mimic DNA) was determined from an
ethidium bromide-stained gel photograph, by densitometry using the
computer program National Institutes of Health Image, version 1.51 (an
updated version of which can be obtained through the Internet at:
http://rsb.info.nih.gov/nih-image).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on HL-60 cell adhesion to HUVEC
Neutrophils and HL-60 cells bind avidly to HUVEC that have been
pretreated with one of a variety of activators, including TNF-,
IL-1ß, but not IFN- (20). As first observed by Hallmann for
neutrophil binding to IL-1ß-activated HUVEC (14), Figure 1
shows that 24-h pretreatment of HUVEC
with IFN- results in significantly reduced HL-60 cell binding to
PMA-activated HUVEC.
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To test whether activation-induced expression of endothelial cell
adhesion molecules was affected by IFN- pretreatment, HUVEC were
pretreated for 24 h with IFN-, then evaluated for the
expression of E-selectin, ICAM-1, VCAM-1, or HLA-DR after 6 h
(Fig. 2A) or 24 h (Fig. 2B) of activation by PMA, TNF-, or IL-1ß. IFN-
treatment alone induced or increased the expression of ICAM-1 and
HLA-DR, and in combination with PMA, TNF-, or IL-1 activation,
IFN- pretreatment further increased the levels of VCAM-1 or ICAM-1.
In contrast, E-selectin expression due to PMA, IL-1, or TNF-
activation was reduced significantly if HUVEC were first pretreated
with IFN- (Fig. 2). This effect was observed both at 6 and 24 h
following addition of activators. The inhibitory effect of IFN- on
activation-dependent E-selectin expression was tested over a wide range
of activator concentrations (Fig. 3).
High concentrations of activators, up to 100 ng/ml PMA, or 25 ng/ml
TNF- or IL-1 did not overcome the inhibitory effect of IFN-
pretreatment on E-selectin induction. The concentrations of IFN-
required to inhibit activation-dependent E-selectin expression were
then compared with the concentrations of IFN- required to induce
ICAM-1 or HLA-DR expression on HUVEC (Fig. 4). Similar concentrations of IFN-
were found to be required to inhibit activation-induced E-selectin
expression and to induce ICAM-1 and HLA-DR expression.
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by anti-IFN- Ab, AF-2
The ability of IFN- pretreatment to inhibit
activation-dependent E-selectin expression by HUVEC was evaluated in
the presence or absence of a neutralizing mAb to IFN-, AF-2 (Fig. 5). In the presence of AF-2, but not an
isotype control mAb, the inhibitory effect of IFN- on PMA-induced
E-selectin expression was reversed, indicating that the effects
observed with IFN- were not due to a nonspecific inhibitor present
in the preparation of IFN-.
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pretreatment effects
To determine the exposure time required for IFN- to inhibit
activation-induced E-selectin expression, HUVEC were treated at the
same time or 1, 2, or 3 days before activation with TNF-, PMA, or
IL-1 for 6 h, then evaluated for E-selectin (Fig. 6A) or ICAM-1 (Fig. 6B) expression. Maximal inhibition of E-selectin expression
induced by TNF-, PMA, or IL-1 requires 1- to 2-day pretreatment
of HUVEC with IFN- (Fig. 6A). If IFN- was added at the
same time as TNF-, PMA, or IL-1 (Fig. 6A, time =
0), equivalent or only slightly reduced levels of E-selectin were
observed when HUVEC were evaluated after 6 h (Fig. 6A).
In contrast, levels of ICAM-1 were similar or slightly enhanced with
IFN- coincubation or 1- to 3-day pretreatment before 6-h activation
with TNF-, PMA, or IL-1 activation (Fig. 6B).
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To evaluate how IFN- pretreatment affects activation-induced
expression of E-selectin on individual cells, flow cytometry was used
to analyze HUVEC pretreated with IFN- (0, 10, or 250 ng/ml) for
24 h, and then activated with PMA for an additional 6 h.
Figure 7 shows that pretreatment of HUVEC
with 250 ng/ml IFN- resulted in reduced levels of E-selectin on
PMA-activated HUVEC and a reduced number of E-selectin-expressing
cells, as both the number of HUVEC expressing E-selectin and the mean
fluorescence intensity of the E-selectin detected were significantly
decreased. Pretreatment of HUVEC with 10 ng/ml IFN-, an amount that
induces an intermediate level of HLA-DR expression on HUVEC, also
reduced the level of E-selectin expression induced by PMA, although to
a lesser extent.
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pretreatment on activation-induced E-selectin
mRNA levels in HUVEC
To evaluate whether IFN- pretreatment affects E-selectin
expression at the mRNA level, the levels of E-selectin mRNA in treated
HUVEC were precisely measured by competitive RT-PCR (Fig. 8) (17, 18, 19). As a control, the levels of
ICAM-1 and GAPDH mRNA were also analyzed. cDNA was prepared from total
RNA of HUVEC pretreated with IFN- (0 or 250 ng/ml) for 48 h and
PMA (100 ng/ml) for an additional 4 h. PCR amplification of native
cDNA (E-selectin, ICAM-1, or GAPDH) was performed in the presence of
various known quantities of modified corresponding cDNA carrying a
short deletion in the amplified region (competitor DNA) as internal
standards (reviewed in 19 . The PCR products from HUVEC-derived
target cDNA (native PCR fragments) and those from exogenously added
competitor DNA (mimic PCR fragments) were separated by agarose gel
electrophoresis and quantified by gel densitometry. When the same
amounts of native and mimic PCR products are produced, the
concentration of competitor DNA is equal to that of target cDNA. Figure 8 (A–C) shows the ratio of native to mimic PCR products at
each concentration of competitor DNA for E-selectin, ICAM-1, and GAPDH.
Based on these results, it is possible to determine the difference in
mRNA levels between the two cDNA samples derived from HUVEC with and
without IFN- pretreatment. After normalization to the mRNA level of
the housekeeping gene GAPDH, the level of ICAM-1 mRNA was found to be
slightly increased (1.6-fold) if HUVEC were first pretreated with
IFN-. On the other hand, pretreatment with IFN- in PMA-activated
HUVEC resulted in >fivefold reduction in the level of E-selectin mRNA
(Fig. 8D).
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on endothelial cell expression of P-selectin
Although PMA, TNF-, and IL-1ß induce expression of E-selectin
on HUVEC, these activators do not up-regulate P-selectin expression on
HUVEC (21). However, very early passage HUVEC do express a constitutive
level of P-selectin that can be increased significantly by IL-4. IL-4
acts at the transcriptional level to increase P-selectin expression,
and unlike E-selectin, which peaks 6 to 8 h following activator
exposure, IL-4-induced P-selectin expression continues to increase and
persist beyond 24 h (21). Since both E- and P-selectin have been
shown to participate in neutrophil recruitment in vivo, the effects of
IFN- on constitutive and IL-4-induced P-selectin expression on HUVEC
were evaluated. Figure 9 shows that
IFN- treatment reduces the level of IL-4-dependent P-selectin
expression on HUVEC to constitutive levels. Pretreatment was not
required, as maximal inhibition of IFN- on IL-4-induced P-selectin
expression was observed when IFN- and IL-4 were added to cultures at
the same time, for 24 h (A), or 48 h
(B). Under the conditions of these assays, a statistically
significant increase in the level of cell surface P-selectin expression
upon 10-min exposure to thrombin could not be detected (data not
shown), in contrast to other studies (21). However, the constitutive
level of cell surface P-selectin expressed by these early passage HUVEC
was not reduced, but slightly increased by IFN- pretreatment (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of cytokines to influence leukocyte-endothelial cell interactions and therefore modulate leukocyte recruitment can be a powerful mechanism through which cytokines control inflammatory and immune responses. While up-regulation of specific adhesion pathways and the subsequent recruitment of select leukocyte subsets are critically important for the inflammatory reactions that protect us from infection, it is equally important for these to be reversed once the infection is eliminated. Tight control of neutrophil recruitment is particularly required, as neutrophils are potent reservoirs of degradative enzymes and reactive oxygen species, that uncontrolled, can mediate significant tissue damage (29). Both TGF-ß and IL-4 have been proposed as factors that down-regulate neutrophil recruitment in vivo, in part through their ability to inhibit the induction of E-selectin on endothelial cells (30, 31). TGF-ß has general antiinflammatory properties and inhibits not only neutrophil, but also mononuclear cell adhesion to endothelial cells in vitro and recruitment in vivo (32, 33, 34). TGF-ß, an important growth factor in wound healing, thus can play a major role in the resolution phase of inflammatory responses (35). IL-4 promotes the recruitment of mononuclear cells through its ability to induce VCAM-1 on endothelial cells (36). The inhibitory effect of IL-4 on neutrophil adhesion, then, has been suggested to be important for the transition to mononuclear cell infiltrate during the progression of inflammatory responses from acute to chronic (30). However, as IL-4 production is typically restricted to Th2-immune responses, the possibility that another cytokine or factor serves this role in Th1-inflammatory responses is intriguing.
Several reports have suggested that the Th1 cytokine IFN- has the
ability to inhibit neutrophil recruitment in vivo (11, 12, 13). After
observing that systemic treatment of mice with IFN- reduced the
number of neutrophils present in thermal wounds, Amento and coworkers
(11) went on to show that the number of neutrophils recruited to
IL-1-treated footpads at 4 h was reduced by 65% if mice were
first systemically treated with IFN-. In another report, rats
treated i.p. with IFN- shifted their response to chronic
Pseudomonas aeruginosa infection in the lung from
neutrophils to mononuclear cells, suggesting a similar effect (13). One
potential mechanism contributing to these results may be related to the
ability of IFN- to up-regulate expression of mononuclear cell
chemoattractants such as monocyte-chemotactic protein-1 and
IFN--inducible 10-kD protein (IP-10), but not
neutrophil-selective chemoattractants such as IL-8 or growth-related
oncogene (GRO) (37, 38, 39). However, the known effects of IFN-
on chemokine expression do not yet explain the inhibitory effect of
IFN- on neutrophil recruitment, as IFN- is not known to
down-regulate production of neutrophil-selective chemokines. On the
contrary, one report describes synergy of IFN- with IL-1 in the
induction of IL-8 by cultured endothelial cells (38).
Studies by Hallmann and coworkers (14) have suggested that an
alternative mechanism by which IFN- can inhibit neutrophil
recruitment may be through modulation of endothelial cell adhesion
molecule expression or function. In these studies, neutrophil adhesion
to IL-1- or TNF--activated HUVEC was reduced significantly if
HUVEC were first pretreated with IFN-. We have confirmed this
observation and extended it by evaluating the activation-dependent
expression of endothelial cell adhesion molecules on HUVEC with and
without IFN- pretreatment. IFN- pretreatment was found to inhibit
the up-regulation of E-selectin expression on HUVEC induced by TNF-,
IL-1, or PMA. Inhibition required IFN- pretreatment and occurred
over the same range of concentrations of IFN- that induce expression
of HLA-DR and ICAM-1. Since both E- and P-selectin can be expressed by
endothelial cells and many experimental systems have demonstrated their
ability to synergize in promoting neutrophil recruitment, it was also
important to evaluate the regulation of P-selectin by IFN-. Although
HUVEC lose the ability to express P-selectin within two to three
passages, primary cultures of HUVEC express a constitutive level of
P-selectin that can be increased by thrombin (at 10 min) or by IL-4 (at
24–48 h) (21). Interestingly, we found that the level of P-selectin
induced by 24- or 48-h activation with IL-4 was reduced significantly
if HUVEC were treated with IFN- at the same time, while the
constitutive level of cell surface P-selectin expression was slightly
increased.
The regulation of endothelial cell E-selectin expression appears to be
exclusively through control of mRNA production and stability (40, 41).
We therefore measured the effect of IFN- on activation-dependent
E-selectin mRNA levels. The ability of IFN- pretreatment of HUVEC to
cause a >fivefold reduction in PMA-induced E-selectin mRNA levels
suggested an effect of IFN- on E-selectin transcription or mRNA
stability. This effect was specific, since the combination of IFN-
pretreatment of HUVEC and PMA activation had little effect on GAPDH or
ICAM-1 mRNA levels. If the effect of IFN- on E-selectin mRNA levels
is on transcription, several hypotheses remain to be evaluated, as the
E-selectin promoter contains multiple regulatory elements, including
three nuclear factor-
B sites, an activating transcription factor-2
(ATF-2) site, and several high mobility group protein-1
(HMG-1) binding sites (reviewed in 40 . Recently, Bennett
et al. (42) proposed an interesting regulatory mechanism through which
IL-4 may inhibit E-selectin expression. They suggest that the
IL-4-induced transcription factor, STAT6, may act as a transcriptional
repressor for E-selectin by binding to the dual nuclear factor-B
enhancer element in the E-selectin promotor. Although IL-4 is a
positive regulator for P-selectin and STAT6 is not known to be induced
by IFN-, other molecules, such as the B cell-specific BCL-6, can
bind to STAT6 sequences and repress transcription (21, 43). Whether or
not transcriptional repression is the mechanism through which IFN-
inhibits activation-induced E-selectin or P-selectin expression, and
whether or not the mechanism for E-selectin is similar or distinct from
the mechanism for P-selectin remain to be studied. Clearly, a large
variety of potential mechanisms are possible, as the molecular
integration of multiple cytokine signals is an area of growing
complexity.
In our experiments, adhesion of HL-60 cells to PMA-activated HUVEC was
reduced 
50% if HUVEC were first preincubated with IFN-. While
such a modest in vitro effect might not be expected to translate in
vivo to a significant attenuation of neutrophil recruitment, it also
must be remembered that physiologically relevant conditions in vivo are
more complex. For example, in most inflammatory situations, multiple
activators are acting on endothelial cells, and in many cases both E-
and P-selectin participate in neutrophil recruitment (44, 45, 46). Thus,
the ability of IFN- to inhibit activation-induced expression of both
E- and P-selectin could be expected to have a more profound impact on
neutrophil recruitment in vivo. It will also be important to evaluate
more carefully the effect of IFN- on the production of
leukocyte-selective chemoattractants in these more complex activating
environments. The ability of IFN- to influence leukocyte recruitment
by acting at multiple steps of the recruitment process, adhesion
molecule expression, and chemoattractant production provides an
attractive mechanism for tight control of a complex process by a single
cytokine.
The findings described in this study can be most easily applied to
situations of in vivo therapy with IFN-. The disparate effects of
IFN- that have been observed in vivo may be explained, in part, by
opposite effects on neutrophil vs mononuclear cell subset adhesion and
recruitment. However, these results also lead to speculation on the
general role of IFN- in inflammatory responses. In the in vitro
studies presented in this work, pretreatment of HUVEC with IFN- was
required for the inhibitory effect on E-selectin expression to be
observed. The requirement for pretreatment may have been simply to
overcome a time lag in the production of a (putative) IFN--inducible
factor responsible for inhibiting E-selectin transcription (or reducing
E-selectin mRNA stability). Expression of E-selectin in vitro is quite
transient, peaking at 4 to 6 h following activation, making
pretreatment a requirement. In vivo, in more physiologic situations,
expression of E-selectin is prolonged. E-selectin is expressed in many
sites of chronic inflammation (47), and in a primate model of cerebral
ischemia/reperfusion injury, E-selectin expression is still maximal at
24-h postischemia (48). In addition, pretreatment was not required for
IFN- to inhibit IL-4-induced P-selectin expression measured at
24 h. It may be that under conditions of prolonged E- and
P-selectin expression, IFN- can play a role in the transition of
inflammatory infiltrates from neutrophilic to mononuclear by modulating
leukocyte adhesion and recruitment mechanisms. Evaluation of such a
role for IFN- awaits further in vivo studies.
Although several groups have examined the effects of IFN- treatment
of HUVEC in combination with TNF- or IL-1ß, the results that we
describe have not been previously reported. In several cases, HUVEC
were treated with IFN- at the same time as TNF- or IL-1ß
activation, not preincubated with IFN- first, as we describe in this
work (36, 49, 50). In another case, although HUVEC were pretreated with
IFN- for 20 h before 5-h activation by TNF- or IL-1ß, a
result opposite from ours was found: increased levels of E-selectin on
HUVEC were observed (51). This opposite result may be due to the
different culture conditions used. In addition, in our experiments we
were careful to verify the activity of the IFN- employed, by
establishing its ability to induce HLA-DR, and by utilizing blocking
mAbs to IFN-. It is also possible that different results can be
obtained with different endothelial cell donors. However, although we
observe donor-dependent differences in the magnitude of IFN-
inhibition of HUVEC E-selectin expression, HUVEC from all donors
evaluated (>10) displayed significant inhibition of E-selectin
induction by IFN- pretreatment at confluent as well as subconfluent
culture conditions. The ability of IFN- to inhibit
activation-dependent E-selectin expression also differed among
activators. We found PMA-induced E-selectin up-regulation to be more
sensitive to IFN- pretreatment than TNF-- or IL-1ß-induced
expression. Interestingly, a recent report describes IFN- inhibition
of dsRNA induced expression of E-selectin (52). The molecular mechanism
for these differences remains to be defined.
Human dermal microvascular endothelial cells apparently behave
differently than HUVEC in regard to E-selectin regulation. Lee and
coworkers (53) report that IFN- induces E-selectin expression on
human dermal microvascular endothelial cells, unlike HUVEC. This may be
related to the observation that cutaneous inflammatory disorders,
characterized by high E-selectin expression on endothelial cells,
contain predominantly cutaneous lymphocyte Ag-positive memory T cells
rather than neutrophils. Thus, tissue-specific differences can also
modify endothelial cell responses to cytokines and other stimuli.
In conclusion, the ability of IFN- to control endothelial cell
adhesion molecule expression and thus regulate leukocyte recruitment is
a powerful mechanism through which IFN- can direct immune and
inflammatory reactions. Novel candidate therapeutics that target
components of this process (cytokines, adhesion molecules, chemokines)
have a promising future for treatment of autoimmune and inflammatory
conditions in which overproduction of IFN- has pathologic
consequences.
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Footnotes |
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2 Abbreviations used in this paper: EGM, endothelial cell growth medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication September 25, 1997. Accepted for publication April 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or medium alone (
for 24 h, and then activated with PMA (100
ng/ml) or media (None) for an additional 4 h. Adhesion of
fluorescently labeled HL-60 cells to treated HUVEC grown in 96-well
plates was evaluated, as described in Materials and
Methods. The relative number of HL-60 cells binding to HUVEC
under the various conditions was determined by measuring the mean
fluorescence intensity (MFI) in each well. The mean ± SD,
n = 4, from a single representative experiment is
shown. **Indicates p < 0.005; NS indicates not
significant, comparing with and without IFN-
