1 Department of Dermatology and
Emory Skin Diseases Research Core Center, There is
increasing evidence that sensory nerves may participate in cutaneous
inflammatory responses by the release of neuropeptides such as
substance P (SP). We examined the direct effect of SP on human dermal
microvascular endothelial cell (HDMEC) intercellular adhesion molecule
1 (ICAM-1) expression and function. Our results indicated that,
although cultured HDMEC expressed mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R), SP initiated a rapid increase in HDMEC
intracellular Ca2+ levels,
primarily by the activation of NK-1R. Immunohistochemistry studies
likewise demonstrated that HDMEC predominantly expressed NK-1R. The
addition of SP to HDMEC resulted in a rapid increase in cellular ICAM-1
mRNA levels, followed by a fivefold increase in ICAM-1 cell surface
expression. This functionally resulted in a threefold increase in
51Cr-labeled binding of J-Y
lymphoblastoid cells to HDMEC. In vivo studies demonstrated a marked
increase in microvascular ICAM-1 immunostaining 24 and 48 h after
application of capsaicin to the skin. These results indicate that
neuropeptides such as SP are capable of directly activating HDMEC to
express increased levels of functional ICAM-1 and further support the
role of the cutaneous neurological system in modulating inflammatory
processes in the skin.
intercellular adhesion molecule 1; cell surface molecules; neuroimmunology; inflammation; skin
UNMYELINATED SENSORY NERVE C fibers that innervate the
skin are capable of releasing neuropeptides such as substance P (SP) after stimulation by injury, pain, temperature, and chemical irritants (3, 20). Although released neuropeptides have been
implicated in the induction of cutaneous inflammation, the mechanisms
by which these effects are mediated are poorly understood. In this study, we examine the possibility that the cutaneous
neurological system may regulate inflammatory responses in the skin
by the direct activation of human dermal microvascular endothelial
cells (HDMEC) and subsequent induction of specific genes
involved in localized inflammatory responses.
A critical component of the initiation and evolution of localized
inflammation is the homing and extravasation of leukocytes at sites of
tissue injury (6). Intercellular adhesion molecule 1 (ICAM-1) is a
90-kDa inducible cell surface glycoprotein that promotes firm adhesion
during leukocyte emigration into sites of inflammation and is
constitutively present at low levels on the endothelial cell surface
(19, 40, 45). We and others have demonstrated that ICAM-1 gene
expression is regulated by a number of cytokines and ultraviolet
radiation (7, 38, 45). Induction of ICAM-1 gene expression is regulated
principally at the level of transcription, and functional analysis of
the ICAM-1 transcriptional regulatory region has implicated multiple
unique pathways by which specific signals induce ICAM-1 gene
transcription (8, 9, 32, 35). In the current study, we examine the direct effect of SP on HDMEC activation as well as ICAM-1 expression and function. SP modulation of HDMEC ICAM-1 expression further supports
the role of the neurological system in mediating cutaneous inflammatory reactions.
Cells and reagents.
HDMEC isolated from foreskins were obtained from the cell culture
facility of the Emory Skin Disease Research Core Center (Atlanta, GA)
(26). Experiments were conducted with cells in passages
3-5.
HDMEC were cultured on a gelatinized (0.1%) surface in MCDB 131 (Life
Technologies, Gaithersburg, MD) supplemented with 10% normal human
serum (Irvine Scientific, Santa Ana, CA), 5 ng/ml epidermal growth
factor (Clonetics, San Diego, CA), 1 mg/ml hydrocortisone acetate
(Sigma Chemical, St. Louis, MO), 5 × 10
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
5 M dibutyryl adenosine
3',5'-cyclic monophosphate (Sigma), 100 U/ml penicillin,
0.25 mg/ml amphotericin B, and 10 µg/ml streptomycin (Life
Technologies). Human B lymphoblastoid J-Y cells (generously provided by
Dr. Jack Strominger, Dana Farber Cancer Institute, Boston, MA) were
cultured in RPMI 1640 supplemented with 10% fetal bovine
serum, 100 U/ml penicillin, 0.25 mg/ml amphotericin B, and 10 µg/ml
streptomycin (Life Technologies).
-Ala8]
neurokinin A 4
10) were obtained from Peninsula Laboratories. An
additional NK-1R antagonist (SR-140333) was generously provided by Dr.
Xavier Emonds-Alt (Sanofi Recherche, Montpellier, France). Lyophilized
human recombinant tumor necrosis factor-
(TNF-
) was obtained from
R & D Systems (Minneapolis, MN).
Determination of neurokinin receptor mRNA expression by RT-PCR. RT-PCR was carried out on untreated HDMEC to measure the expression of NK-1R, NK-2R, and NK-3R mRNA. HDMEC were expanded in culture to 1 × 106 cells, and mRNA was harvested via an mRNA isolation kit according to the manufacturer's instructions (Boehringer Mannheim, Indianapolis, IN). First-strand cDNA was first synthesized using 0.5 µg of oligo(dT)15 as primer, 1 mM dNTPs, 15 units of Moloney murine leukemia virus RT, and 1 µg of mRNA in a reaction volume of 20 µl, according to the manufacturer's recommended instructions for the Promega reverse transcription system (Promega, Madison, WI). One-twentieth of this reaction product was then subjected to PCR template. Oligonucleotide primers were utilized to amplify a portion of each neurokinin receptor cDNA based on the published sequences (5, 12, 15). The primer sequences were, for NK-1R, 5'-CCTGCTGGTGATTGGCTATG-3' (sense) and 5'-CTCTGGCTCCTCCTCGTGGG-3' (antisense); for NK-2R, 5'-GCCTCCCCTCAGTGCTTCTA-3' (sense) and 5'-GGGGGCAAGCAAACCATACC-3' (antisense); and for NK-3R, 5'-TTCTACTTGCCTTCCCTCAG-3' (sense) and 5'-GTTGCTCTTTTCTTCCGACT-3' (antisense). The predicted PCR amplification products of 487, 687, and 655 bp were generated from HDMEC cDNA templates as fragments of NK-1R, NK-2R, and NK-3R, respectively. The amplification profile involved a step for denaturing of the template with 1× PCR buffer for 5 min at 95°C, followed by the addition of 200 µM each dNTP, 1.0 µM each primer, 0.5 U/100 µl Taq polymerase, and 1 pmol/µl sense and antisense primers with 1× PCR buffer and then 35 cycles of amplification, each composed of denaturation at 94°C for 10 s, primer annealing for 10 s (at 58.1°C for NK-1R, 59.3°C for NK-2R, and 53.1°C for NK-3R), and polymerase extension at 72°C for 10 s.
Measurement of intracellular
Ca2+.
Cultured HDMEC were detached by a brief exposure to cell dissociation
buffer, an enzyme-free PBS-based buffer (Life Technologies). Cells were
then washed twice with PBS() (no Ca2+ or
Mg2+), and fura 2-AM (Molecular Probes, Eugene, OR) was
incorporated into 3-5 × 107 cells by its addition to 5 ml
of cell suspension at a final concentration of 5-10 µM and
incubated for 45 min at 37°C. After three washes with PBS(
)
buffer, ~6 × 106 cells/ml
were transferred to a quartz cuvette and stirred continuously. The
cells were then treated with SP alone or were pretreated for 20 min
with 1,000 nM neurokinin receptor antagonists (NK-1R, NK-2R, or NK-3R
antagonist, Peninsula Laboratories), followed by treatment with 100 nM
SP. The neurokinin receptor antagonists were used singly or in
combination, as indicated. Fluorescence was recorded in a model LS50
spectrofluorometer (Perkin-Elmer, Branchburg, NJ), with excitation
wavelengths of 340 and 380 nm and an emission wavelength of 510 nm.
From the ratio of measured fluorescence at the two excitation
wavelengths, the intracellular free
Ca2+ concentration was calculated
as described (39). As controls for each experiment, fluorescence was
also measured after treatment of the cell suspension with Triton X-100
(0.3%) to lyse the cells and generate maximum
Ca2+ flux and after treatment with
EGTA (10 nM) to chelate Ca2+ and
abrogate the response.
Determination of in vivo neurokinin receptor expression in
microvascular cells by immunohistochemistry.
Primary antibodies were raised in rabbits to peptide fragments of
NK-1R, NK-2R, and NK-3R conjugated to keyhole limpet hemocyanin, as
recently described (14). Punch biopsies obtained from normal skin of
volunteers were immediately frozen in liquid nitrogen, embedded in
Tissue-Tek OCT compound (Miles), and stored at 80°C. Frozen
sections were cut 5-7 µm thick on a cryostat, mounted on Superfrost slides (Fisher Scientific, Pittsburgh, PA), fixed for 20 min
in 4% paraformaldehyde at room temperature, and washed in PBS.
Endogenous peroxide was blocked with
H2O2
(1%) in methanol solution for 30 min. Slides were pretreated with 5%
normal rabbit serum, 1% BSA, and 0.3% Triton X-100 for 30 min. After
a washing in PBS, primary antisera were applied (NK-1R, 1:1,000 to
1:3,000; NK-2R, 1:2,000 to 1:4,000; NK-3R, 1:1,000) in
PBS, 1% normal goat serum, and 0.3% Triton X-100 and
incubated for 12 h at 4°C in a humidified chamber. Immunoreaction
was visualized using the unlabeled antibody-enzyme streptavidin-biotin
complex (Vectastain, Vector Laboratories) technique, as previously
described (22). After thorough rinsing of slides with PBS, the
secondary antibody (1:100 in PBS-0.25% BSA) was applied for 30 min at
room temperature. After a washing, the streptavidin-peroxidase complex
was added at a dilution of 1:200 for 30 min at room temperature.
Finally, staining was visualized by incubation in a solution containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01%
H2O2
in Tris-buffered saline (pH 7.6). Slides were dehydrated in graded
alcohols and xylene and mounted in Paramount medium (Fisher
Scientific). To control for specificity of immunodetection, antisera
were incubated with an excess of the corresponding peptides (10
5 M) used for
immunization and utilized in immunohistochemistry of frozen tissue
sections as above (14). As a positive control for each antibody,
immunohistochemistry was performed using monolayers of Kirsten murine
sarcoma virus-transformed rat kidney cells transfected with the gene
for NK-1R or using Chinese hamster ovary (CHO) cells transfected with
the NK-2R gene or NK-3R gene (34, 47).
Determination of ICAM-1 mRNA expression by Northern blot analysis.
Cultured HDMEC (1 × 106
cells) were treated with 100 nM SP for 1, 3, or 5 h or with 30 U/ml
TNF- for 5 h. TNF-
is utilized as a positive control for ICAM-1
induction on HDMEC. Total cellular mRNA was isolated, and Northern blot
analysis was done as previously described (9). The ICAM-1 probe
consists of a 0.67-kb Stu I fragment
of the human ICAM-1 cDNA (generously provided by Dr. D. E. Staunton, Dana Farber Cancer Institute) (44). Hybridization with
radiolabeled
-actin cDNA was used for determination of lane-loading consistency and for normalization of ICAM-1 signal under the various conditions tested. The autoradiograph was scanned on a La Cie flatbed
scanner (La Cie, Beaverton, OR) utilizing Adobe Photoshop software
(Adobe Systems, Mountain View, CA). Subsequently, the digitized image
was analyzed using NIH Image software (National Institutes of Health,
Bethesda, MD), and the relative intensity was calculated as the ratio
of pixels per square inch of ICAM-1 to
-actin per lane.
Determination of HDMEC surface expression of ICAM-1 by ELISA.
HDMEC were plated onto 96-well plates and on reaching confluence were
stimulated with various concentrations of SP for fixed time periods or
with a fixed concentration of SP for various time periods. TNF- at
300 U/ml was used as a positive control for ICAM-1 cell surface
expression. For ICAM-1 dose response studies, HDMEC were stimulated
with SP at 10, 100, or 1,000 nM for 18 h. For ICAM-1 induction kinetics
studies, HDMEC were stimulated with SP at 100 nM for 1-24 h. After
the stimulation period, HDMEC cell surface ICAM-1 expression was
assessed by a two-step ELISA as previously described (9), using the
mouse anti-human ICAM-1 monoclonal antibody (MAb) 84H10 followed by a
peroxidase-conjugated goat anti-mouse IgG that was detected by a
3,3',5,5'-tetramethylbenzidine colorimetric reaction and
read as optical density at 450 nm. Results are means ± SD of four
values for each variable tested, and results are representative of
three independent assays.
Determination of surface ICAM-1 expression on HDMEC by flow
cytometric analysis.
For flow cytometric analysis, cultured HDMEC were stimulated with 10 or
100 nM SP for 18 h. In selected experiments, cells were pretreated with
the specific NK-1R antagonist SR-140333 (1,000 nM) for 20 min before
the addition of 100 nM SP. HDMEC were detached from tissue culture
plates with 0.05% trypsin-0.53 mM EDTA (Life Technologies) and washed
with PBS() (wash buffer), and aliquots were transferred into
tubes for antibody staining. HDMEC were incubated with mouse anti-human
ICAM-1 MAb 84H10, or isotype control mouse anti-human IgG heavy and
light chains (H+L) (Jackson Immunoresearch, West Grove,
PA) at a final concentration of 10 µg/ml for 1 h on ice. Cells were
washed twice and incubated with FITC-conjugated affinity-purified goat
F(ab')2 anti-mouse IgG (H+L)
(Jackson Immunoresearch) at a final concentration of 10 µg/ml for 1 h
on ice. Subsequently, cells were washed twice and analyzed by a FACscan
flow cytometer (Becton Dickinson, Raleigh, NC) equipped with CellQuest
software for data acquisition and analysis. The forward scatter
threshold was set to permit analysis of viable endothelial cells.
Adherence of J-Y lymphoblastoid cells to HDMEC.
Binding assays measuring cellular adherence to HDMEC were performed
with human lymphoblastoid J-Y cells that preferentially express the
ICAM-1 ligand, leukocyte function-associated antigen-1 (LFA-1), and
consequently adhere to ICAM-1 on target cells (10). HDMEC were plated
in 24-well plates overnight and then either left untreated or treated
for 18 h with increasing doses of SP or 300 U/ml TNF- diluted in
HDMEC assay medium in triplicate wells, and then plates were washed
three times with Hanks' balanced salt solution plus
(HBSS+, with Ca2+ and
Mg2+). In selected control and treated
wells, anti-ICAM-1 MAb 84H10 or anti-vascular cell adhesion molecule 1 (VCAM-1) MAb P3C4 (generous gift of Dr. Elizabeth Wayner, University of
Minnesota, Minneapolis, MN) were added at a final concentration of 10 µg/ml and allowed to incubate for 10 min at 37°C in 5%
CO2. J-Y cells were pelleted, resuspended in HBSS+, and
incubated with 500 µCi of 51Cr
for 1 h at 37°C in 5% CO2.
The labeled J-Y cells were washed twice with
HBSS+ and then added to control
and variously treated HDMEC wells at a concentration of 70,000 cells/well and incubated for 20 min at 37°C. The supernatant was
removed, and the J-Y-overlayed HDMEC were washed gently three times
with HBSS+ to remove nonadherent
J-Y cells. SDS (1%) was added and allowed to incubate for 15 min at
room temperature. Each well was then swabbed with two cotton-tipped
swabs that were counted in a gamma counter. Adherence was calculated as
%J-Y binding = [cpm per well
background cpm] /
[cpm added counts
background cpm] × 100, as
previously described (42), where cpm is counts/min. To photograph J-Y
cell adhesion to SP-treated HDMEC, unlabeled J-Y cells were added
according to the same protocol, stopping before the addition of SDS.
Photomicrographs were taken using Kodak Ektachrome 66 tungsten film on
an Olympus OM-2 camera mounted on an Olympus CK2 microscope.
Determination of in vivo expression of ICAM-1 in human skin
microvascular cells by immunohistochemistry.
Capsaicin (Zostrix, 0.075%, GenDerm, Lincolnshire, IL) was applied
topically to the skin of a volunteer to stimulate the release of
cutaneous neuropeptides, and 4-mm punch biopsies were taken from
treated sites at 6, 24, and 48 h. Biopsies were also taken from an
untreated site on the opposite limb immediately before capsaicin
application. Tissue was embedded in OCT and frozen at 70°C.
Immunohistochemistry was performed on 8-µm sections using mouse
anti-human ICAM-1 MAb 84H10 as the primary antibody (diluted 1:400), a
biotinylated horse anti-mouse secondary antibody (Zymed), and
streptavidin-biotin complex (Zymed) as a tertiary reagent to detect
specific binding of the primary and secondary reagents. Samples were
examined using a Nikon Microshot SA microscope and photographed using a
Nikon NFX-35 microscope camera. Control staining was performed on
tissue processed without the primary antibodies. No staining was
obtained with either of the controls.
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RESULTS |
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HDMEC express mRNA for neurokinin receptors. SP is capable of specifically binding to NK-1R, NK-2R, and NK-3R with high, intermediate, and low affinity, respectively (3, 18). The expression of these receptors was examined in HDMEC by nonquantitative RT-PCR. As demonstrated in Fig. 1, HDMEC expressed mRNA for NK-1R (487 bp), NK-2R (687 bp), and NK-3R (655 bp). Thus HDMEC express the mRNAs for the three neurokinin receptors capable of binding SP.
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SP induces intracellular Ca2+ increase in HDMEC. Neurokinin receptors are G protein-linked receptors that trigger a rapid increase in intracellular Ca2+ when activated (25). To determine whether HDMEC are capable of responding to SP, we examined the ability of SP to induce intracellular Ca2+ influx and mobilization in these cells (Fig. 2). Treatment of HDMEC with 100 nM SP resulted in an immediate and transient increase in intracellular Ca2+ (Fig. 2). The effect of specific antagonists was then examined on SP induction of HDMEC intracellular Ca2+ responses (Fig. 2). As indicated, pretreatment of cells with antagonists for NK-1R, NK-2R, and NK-3R for 20 min before application of SP completely abrogated the intracellular Ca2+ response. In contrast, pretreatment of cells with the NK-3R antagonist alone had little effect on SP intracellular Ca2+ responses, whereas the NK-2R antagonist was capable of partially blocking this response. Importantly, treatment of cells with NK-1R antagonist alone was capable of completely blocking the SP activation of HDMEC. These data suggest that SP primarily activates HDMEC through NK-1R but that some cross-reactivity of the NK-1R antagonist for NK-2R may exist. Treatment of the cells with Triton X-100 followed by EGTA showed the expected increase in Ca2+ mobilization and subsequent return to baseline. Thus SP is capable of specifically inducing Ca2+ influx and mobilization in HDMEC, an activation signal that could result in the induction of downstream events associated with an inflammatory response. This effect appears to be mediated primarily by the activation of the HDMEC NK-1R.
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Microvascular cells express NK-1R in vivo. Neurokinin receptors were immunolocalized in normal human skin using specific antisera to NK-1R, NK-2R, and NK-3R (14). There was significant NK-1R immunostaining of microvascular cells in the dermis (Fig. 3A; see also Fig. 4), but weak immunostaining of these cells was observed with antisera for NK-2R (Fig. 3B). No immunostaining was observed using antisera for NK-3R (data not shown). No immunoreaction was seen when antisera were preabsorbed with an excess of either NK-1R peptide (Fig. 3C) or NK-2R peptide (Fig. 3D), which demonstrates the specificity of the immunostaining. Positive immunostaining was observed using control Kirsten rat kidney cells or CHO cells transfected to express each neurokinin receptor (34, 47) and stained with the respective antisera for that receptor (data not shown). Taken together, these data suggest that the predominant cell surface functional neurokinin receptor on HDMEC in vitro and microvascular endothelial cells in vivo is NK-1R.
|
SP induces ICAM-1 mRNA expression in HDMEC.
To ascertain whether SP is capable of modulating ICAM-1 mRNA in HDMEC,
Northern blot analysis was performed (Fig.
4A).
HDMEC were treated with 100 nM SP for 1, 3, or 5 h or with 30 U/ml
TNF-. HDMEC ICAM-1 mRNA expression was normalized to
-actin mRNA
expression by densitometric analysis for each experimental condition
(Fig. 4B). As indicated, HDMEC constitutively
expressed low levels of ICAM-1 mRNA, which increased 1.25-fold 1 h
after treatment with SP. ICAM-1 mRNA levels further increased 2.5-fold
3 h after the addition of SP but decreased to constitutive mRNA
expression levels 5 h after the addition of SP. TNF-
induction of
HDMEC ICAM-1 mRNA expression served as a positive control for this
study. Thus SP is capable of inducing ICAM-1 mRNA expression in
HDMEC.
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SP induces cell surface ICAM-1 expression on HDMEC. To determine whether increased HDMEC ICAM-1 mRNA expression after SP treatment results in increased ICAM-1 cell surface expression, cells were treated with various concentrations of SP for 18 h, and ICAM-1 expression was measured by ELISA. As indicated in Fig. 5A, SP upregulated ICAM-1 cell surface expression in a concentration-dependent fashion. A fourfold increase in HDMEC ICAM-1 cell surface expression over constitutive levels was observed after treatment with 10 nM SP, and a fivefold increase in ICAM-1 expression was observed after treatment with 100 nM SP (Fig. 5A); 1,000 nM SP was only able to induce a small increase in ICAM-1 expression. We (24) and others (16) have observed dose responses in other neuropeptide studies. Thus 100 nM SP would appear to be the optimal concentration for the induction of cell surface HDMEC ICAM-1.
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SP enhances binding of J-Y lymphoblastoid cells to HDMEC.
The functional consequences of SP induction of cell surface ICAM-1 on
binding of J-Y lymphoblastoid cells to HDMEC were determined using a
quantitative cellular adhesion assay (10). Although 51Cr-labeled J-Y cells showed
minimal binding to untreated cultured HDMEC, the adhesion of J-Y cells
to SP-treated HDMEC increased in a concentration-dependent
manner following the addition of 10 or 100 nM SP for 18 h (Fig.
7). As indicated, 100 nM SP induced a
threefold increase in adhesion of J-Y cells to HDMEC. There was little
or no increase in J-Y cell adhesion to HDMEC treated with 1,000 nM SP,
reflecting our previous studies that demonstrated a poor ICAM-1 cell
surface inductive response after this high concentration of SP (data
not shown). Contribution of ICAM-1 to the adherence of J-Y cells to
HDMEC after SP treatment was examined. Leukocyte binding to SP-treated
HDMEC could be prevented by pretreatment of HDMEC with an anti-ICAM-1
blocking antibody before the addition of labeled J-Y cells (Fig. 7).
The anti-ICAM-1 antibody alone, or an anti-VCAM-1 antibody used as an
irrelevant antibody (data not shown), had little effect on J-Y cell
binding to HDMEC. TNF- treatment of HDMEC served as a positive
control for induction of J-Y binding.
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In vivo ICAM-1 induction in capsaicin-treated human skin. The effect of in vivo release of cutaneous neuropeptides on dermal endothelial cell ICAM-1 expression was examined. A capsaicin-containing cream (Zostrix) was applied to the skin of a human volunteer to stimulate the release of neuropeptides, including SP from cutaneous sensory nerves (31). Biopsies were obtained at 0, 6, 24, and 48 h and were evaluated by immunohistochemistry for ICAM-1 expression associated with HDMEC. Low constitutive levels of microvascular ICAM-1 immunoreactivity were observed in untreated skin (Fig. 9a), and endothelial cell ICAM-1 expression gradually increased at 6 h post-capsaicin application (Fig. 9b). A marked increase in HDMEC ICAM-1 staining was observed by 24 h after application of topical capsaicin (Fig. 9c), and the increase remained 48 h after capsaicin application (Fig. 9d). Thus the release of neuropeptides by cutaneous sensory nerves results in increased in vivo microvascular endothelial ICAM-1 expression.
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DISCUSSION |
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There is increasing evidence that skin inflammation can be mediated by
the cutaneous neurological system through the release of neuropeptides,
such as SP, that interact with target skin cells. The close physical
association of cutaneous nerves with target cells has been established
(21, 49, 50). SP has been well characterized as a potent vasodilator
that can cause increased microvascular permeability and protein
extravasation (11). Additionally, SP can induce leukocyte effector
activities, such as lymphocyte proliferation, cytotoxicity, and
immunoglobulin production (3, 36); mast cell degranulation (37);
macrophage and polymorphonuclear leukocyte activation (17, 51); and
cytokine production by monocytes (23, 28, 30). Our previous studies
indicate that SP can directly activate mast cells and keratinocytes to
secrete TNF- and interleukin-1 (IL-1), respectively (2, 4).
The assessment of cutaneous neuropeptide modulation of HDMEC adhesion
molecule expression is important to our understanding of interactions
between the cutaneous neurological system and skin inflammatory
responses. Inflammatory infiltration of leukocytes into all tissues,
including the skin, depends on leukocyte passage into tissue from the
microvasculature in a multi-step binding interaction involving cell
adhesion molecules expressed by leukocytes and endothelial cells (27,
48). P- and E-selectins mediate initial leukocyte adhesion to HDMEC
through binding to specific carbohydrate moieties present on
neutrophils, whereas ICAM-1 and VCAM-1 mediate subsequent, firm
adhesion and transendothelial migration through binding to
2- and
1-integrins on leukocytes. In
the skin, HDMEC expression of ICAM-1 is an essential component of
cutaneous inflammation. We have previously shown that ICAM-1 is
constitutively expressed in vitro by HDMEC and that the proinflammatory cytokines interferon-
, TNF-
, and IL-1, as well as ultraviolet B
radiation, increase ICAM-1 surface expression (7, 45). The direct
effect of neuropeptides on HDMEC ICAM-1 expression has not been
previously examined.
Our present evaluation of the effect of SP on cutaneous microvascular endothelial cell ICAM-1 expression significantly expands our understanding of the role of the cutaneous neurological system in inflammatory responses in the skin. Our results indicate that cultured HDMEC express mRNA for NK-1R, NK-2R, and NK-3R, which are capable of binding to SP with high, intermediate, and low affinity, respectively. We demonstrate that SP induces a rapid intracellular Ca2+ response in HDMEC and that this effect is mediated primarily by NK-1R. In vivo, immunohistochemistry studies show that NK-1R is the major neurokinin receptor expressed on dermal microvascular cells. HDMEC activation is further accompanied by increased levels of ICAM-1 mRNA and ICAM-1 cell surface expression. In parallel, increased leukocyte binding is observed in SP-treated HDMEC. A similar induction of HDMEC ICAM-1 expression is observed in vivo after the topical application of the SP-releasing agent capsaicin.
Our investigation of the role of microvascular endothelial ICAM-1 upregulation in neurogenic inflammation is supported by several prior studies that suggest neuropeptides may be capable of influencing adhesion molecule expression on these target cells in the skin. We believe that our study is the first to definitively demonstrate that SP is capable of directly activating HDMEC to express increased levels of functional ICAM-1 in vitro and in vivo. In a previous study, Nakagawa et al. (33) reported that SP can upregulate ICAM-1 expression in vitro on large-vessel human umbilical vein endothelial cells. However, this study did not evaluate the presence and function of neurokinin receptors on these cells or the functional consequences of ICAM-1 upregulation, nor was in vivo correlation examined. Because all tissue inflammatory responses in the skin and elsewhere are mediated by microvascular endothelial cell activities rather than large-vessel endothelial cells and because the two cell types display distinct differences in their phenotypes and responses to proinflammatory signals (46), we believe that it is critical to examine HDMEC when studying neuroinflammation responses in the skin.
Some prior studies have utilized murine models to begin to evaluate adhesion molecule expression following administration of neuropeptides. Saban et al. (41) focused on the responses of neutrophils and eosinophils to SP following injection in mouse skin. ICAM-1 involvement was indirectly demonstrated in terms of the ability of pretreatment of mice with MAb to ICAM-1 to inhibit SP-induced leukocyte migration, but there was no determination of ICAM-1 expression on endothelial cells. Goebeler et. al. (13) examined the effect of SP and another neuropeptide, calcitonin gene-related peptide (CGRP), on leukocyte infiltration during allergic contact dermatitis in mouse skin. These authors measured increased ICAM-1 on tissue macrophages and did not observe an upregulation of ICAM-1 on SP-treated murine endothelial cell lines in vitro.
Using an in vivo human skin model system, Smith et. al. (43)
demonstrated that injection of the neuropeptides SP, vasoactive intestinal polypeptide, and CGRP into normal human skin mediated a
rapid accumulation of neutrophils. Using immunohistochemistry, these
investigators noted a parallel upregulation of P-selectin and
E-selectin over an 8-h time period, but constitutive ICAM-1 expression
on dermal endothelium was unchanged at these early time points after
neuropeptide injection. These findings are consistent both with the
expected time course of ICAM-1 upregulation (6, 7) and with the results
of our current study, which indicate that microvascular ICAM-1
immunoreactivity was significantly increased over constitutive levels
at 24 and 48 h after treatment with capsaicin. Murphy and colleagues
(29) have demonstrated that SP treatment of skin explants caused
mast-cell degranulation and subsequent TNF--mediated induction of
E-selectin on postcapillary venular endothelial cells. In
contrast to these findings, we observed that SP was capable of directly
inducing increased HDMEC ICAM-1 expression without the participation of
mast cells. In vivo, both processes are likely to play a role.
In summary, our studies demonstrate that SP is capable of directly regulating HDMEC ICAM-1 expression and function by the activation of specific neurokinin receptors. These findings further support the role of neuropeptide modulation of leukocyte recruitment in the skin during cutaneous inflammatory reactions. The coordination of signals such as neuropeptide release and adhesion molecule upregulation may play a role in the development of a wide range of inflammatory responses and during wound healing. We also have recent evidence that SP is capable of specifically inducing another adhesion molecule, VCAM-1, on HDMEC, which further suggests that this neuropeptide may be involved in multiple inflammatory responses in the skin (1). Further investigation of the interactions of various neuropeptides, adhesion molecules, and other inflammatory mediators in the skin should provide a rational basis for formulating an effective, specific approach to the control of neurogenic inflammation. Such approaches could lead to novel therapies that have application to a wide range of inflammatory processes, such as psoriasis, atopic eczema, and wound healing.
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ACKNOWLEDGEMENTS |
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We thank Marcia Usui for technical expertise in performing immunohistochemistry studies and Neera Bahl (Emory Skin Disease Research Core Center) for assistance in the isolation of human foreskin microvascular endothelial cells.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HD-33024 (to J. Ansel, N. Bunnett, and J. Olerud), AI-41493 (to J. Ansel and S. W. Caughman), AR-41206 and AR-42687 (to S. W. Caughman), and DK-43207 and DK-39957 (to N. Bunnett) and by a Department of Veterans Affairs Merit Review Award (to J. Ansel).
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. §1734 solely to indicate this fact.
Address for reprint requests: J. C. Ansel, Dept. of Dermatology, Emory University School of Medicine, 5001 Woodruff Memorial Bldg., Atlanta, GA 30322.
Received 26 June 1998; accepted in final form 26 August 1998.
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