Neuropeptide regulation of human dermal microvascular endothelial cell ICAM-1 expression and function

Kimberly L. Quinlan1, In-Sung Song1, Nigel W. Bunnett2, Eleanor Letran3, Martin Steinhoff2, Brad Harten1, John E. Olerud3, Cheryl A. Armstrong1,4, S. Wright Caughman1, and John C. Ansel1,4

1 Department of Dermatology and Emory Skin Diseases Research Core Center, Emory University School of Medicine and 4 Department of Veterans Affairs Medical Center, Atlanta, Georgia 30322; 2 Departments of Physiology and Surgery, University of California, San Francisco, California 94140; and 3 Department of Dermatology, University of Washington, Seattle, Washington 98195

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 × 10-5 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).

Lyophilized SP (Peninsula Laboratories, Belmont, CA) was diluted in the appropriate volume of HDMEC assay medium immediately before use. In selected studies, SP receptor antagonists were added to cells 30 min before the addition of SP. Neurokinin receptor 1 (NK-1R) antagonist (GR-82334), neurokinin receptor 2 (NK-2R) antagonist (GR-94800), and neurokinin receptor 3 (NK-3R) antagonist ([Trp7,beta -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-alpha (TNF-alpha ) 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-alpha for 5 h. TNF-alpha 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 beta -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 alpha -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-alpha 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-alpha 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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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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Fig. 1.   Human dermal microvascular endothelial cells (HDMEC) express mRNA for neurokinin receptors 1, 2, and 3 (NK-1R, NK-2R, and NK-3R). RT-PCR products were amplified from HDMEC mRNA using RT-generated cDNA template and paired primers for neurokinin receptors. PCR amplification products of 487, 687, and 655 bp represent specific fragments of NK-1R, NK-2R, and NK-3R, respectively. Labeled standard lanes indicate that amplification products were of expected sizes. Data are representative of experiments conducted in triplicate.

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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Fig. 2.   Substance P (SP) induces intracellular Ca2+ influx and mobilization in HDMEC by activation of specific neurokinin receptors. Cultured HDMEC were suspended in PBS, loaded with fluorescent Ca2+ probe fura 2-AM, and then treated with 100 nM SP with or without pretreatment with 1,000 nM neurokinin receptor antagonists alone or in combination, as indicated, for 20 min. NK-1RA, NK-2RA, and NK-3RA are antagonists of NK-1R, NK-2R, and NK-3R, respectively (see Cells and Reagents). Intracellular free Ca2+ concentration was determined from ratio of measured fluorescence at excitation wavelengths of 340 and 380 nm. Treatment of cells with Triton X-100 (0.3%) led to maximal Ca2+ flux, and treatment with EGTA (10 nM) abrogated response. These results are representative of experiments conducted in triplicate and are depicted as measured 340-nm-to-380-nm excitation ratio vs. time.

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.


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Fig. 3.   Microvascular cells express NK-1R in vivo. Photomicrographs were taken of human skin biopsies immunostained for expression of neurokinin receptors using NK-1R antisera (A and C) or NK-2R antisera (B and D). Arrows, dermal microvascular structures. Specificity of staining was demonstrating by preabsorbing antisera with respective neurokinin receptor peptides before immunostaining (C and D).

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-alpha . HDMEC ICAM-1 mRNA expression was normalized to beta -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-alpha 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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Fig. 4.   SP induces HDMEC intercellular adhesion molecule 1 (ICAM-1) mRNA. Effect of SP on HDMEC ICAM-1 was measured by Northern blot analysis. A: constitutive ICAM-1 mRNA expression (-) was compared with HDMEC treated with 100 nM SP for 1, 3, and 5 h or with 30 U/ml tumor necrosis factor-alpha (TNF-alpha ). B: relative intensity of ICAM-1 expression was determined by densitometry as ratio of pixels per square inch of ICAM-1 and beta -actin mRNA per lane. Data are representative of experiments conducted in triplicate.

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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Fig. 5.   ELISA measurement of ICAM-1 expression of SP-treated HDMEC. A: HDMEC were stimulated with 10, 100, and 1,000 nM SP or 300 U/ml TNF-alpha at 37°C for 18 h, and cell surface ICAM-1 was measured by ELISA. B: HDMEC were also stimulated with 100 nM SP for 1-24 h or with 300 U/ml TNF-alpha for 18 h at 37°C, and cell surface expression was measured by ELISA. Results are means ± SD of 4 values. * Statistically significant differences in cell surface ICAM-1 in treated samples compared with untreated control cells (-) as determined by Student's t-test (P < 0.005). Data are representative of experiments conducted in triplicate.

The kinetics of this SP-ICAM-1 response were also examined. Using a fixed concentration of 100 nM SP, maximum induction of HDMEC ICAM-1 expression was observed between 16 and 18 h after stimulation with SP, inducing a fivefold increase in cell surface ICAM-1 expression compared with untreated HDMEC (Fig. 5B). TNF-alpha induction of HDMEC ICAM-1 served as a positive control in these studies. These data demonstrate that, in addition to the induction of HDMEC ICAM-1 mRNA, SP upregulates the expression of cell surface ICAM-1 on HDMEC in a concentration- and time-dependent fashion.

The effect of SP on HDMEC ICAM-1 cell surface expression was also examined by flow cytometric analysis. Untreated HDMEC expressed low constitutive levels of surface ICAM-1 (Fig. 6A). As in the ELISA studies, treatment of HDMEC with 10 and 100 nM SP for 18 h resulted in a significant concentration-dependent increase in cells expressing surface ICAM-1 compared with control untreated cells (Fig. 6A, bottom left and top right). TNF-alpha -treated HDMEC demonstrated high levels of cell surface ICAM-1 expression and served as a positive control (Fig. 6A, bottom right). First-step incubation with an irrelevant isotype-matched control antibody demonstrated the specificity of the ICAM-1-detected signal seen after SP and TNF-alpha (Fig. 6A, top left).


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Fig. 6.   Flow cytometric analysis of ICAM-1 expression of SP-treated HDMEC. A: surface expression of ICAM-1 on HDMEC was assessed by flow cytometric analysis 18 h after exposure to 10 nM SP (bottom left), 100 nM SP (top right), or 300 U/ml TNF-alpha (bottom right). B: surface expression of ICAM-1 on HDMEC was also assessed by flow cytometric analysis 18 h after exposure to NK-1RA followed by 100 nM SP (bottom left), 100 nM SP (top right), or 300 U/ml TNF-alpha (bottom right). Open histogram areas under solid lines represent constitutive ICAM-1 expression (Untx Ctrl); superimposed filled histogram areas represent expression with treatments indicated by heavier solid lines in keys. In A and B, top left shows untreated cells incubated with an isotype control IgG in place of anti-ICAM-1 antibody. Data are representative of experiments conducted in triplicate.

As indicated in Fig. 6B, pretreatment of HDMEC with an NK-1R antagonist followed by the addition of 100 nM SP blocked the SP induction of cell surface ICAM-1 nearly to the level of constitutive ICAM-1 expression of untreated cells (Fig. 6B, bottom left). Treatment of HDMEC with 100 nM SP alone resulted in a significant increase in cells expressing surface ICAM-1 (Fig. 6B, bottom left and top right). TNF-alpha -treated HDMEC again served as a positive control (Fig. 6B, bottom right), and first-step incubation with an irrelevant isotype-matched control antibody demonstrated the specificity of the ICAM-1-detected signal (Fig. 6B, top left).

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-alpha treatment of HDMEC served as a positive control for induction of J-Y binding.


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Fig. 7.   Increased binding of 51Cr-labeled J-Y lymphoblastoid cells to SP-treated HDMEC. Functional binding of SP-induced ICAM-1 on HDMEC was determined in a quantitative cell adhesion assay utilizing J-Y lymphoblastoid cells. HDMEC were treated for 18 h with 10, 100, and 1,000 nM SP or 300 U/ml TNF-alpha , followed by addition of 51Cr-labeled J-Y cells. Plates were washed, and adherent cell binding activity was expressed as %J-Y binding. Specificity of ICAM-1-mediated binding was determined by pretreatment with anti-ICAM-1 antibody (alpha -ICAM-1) of both untreated HDMEC and SP-treated HDMEC before incubation with J-Y cells. * Statistically significant differences in %J-Y binding in treated samples compared with untreated control cells (-) as determined by Student's t-test (P < 0.005). Data are representative of experiments conducted in triplicate.

The ability of SP to augment J-Y cell binding to HDMEC was also visually demonstrated in photomicrographs of the cellular adhesion assay (Fig. 8). Untreated HDMEC showed minimal numbers of adherent J-Y cells (Fig. 8A), whereas HDMEC treated with 100 nM SP showed high numbers of adherent J-Y cells (Fig. 8B). Again, adhesion of J-Y cells to HDMEC could be blocked by the addition of the anti-ICAM-1 antibody to the SP-treated cells (Fig. 8C). As expected, large numbers of J-Y cells adhered to HDMEC monolayers pretreated with TNF-alpha (Fig. 8D). These data demonstrate that SP is capable of augmenting ICAM-1-dependent binding of LFA-1-expressing lymphoblastoid cells to HDMEC.


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Fig. 8.   Photomicrographs of J-Y lymphoblastoid cell binding to SP-treated HDMEC. Photomicrographs were taken of J-Y lymphoblastoid cells binding to untreated HDMEC (A), HDMEC treated with 100 nM SP (B), HDMEC treated with 100 nM SP followed by anti-ICAM-1 antibody (C), and HDMEC treated with 300 U/ml TNF-alpha (D). Data are representative of experiments conducted in triplicate.

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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Fig. 9.   In vivo induction of HDMEC ICAM-1 expression. Photomicrographs were taken of human skin biopsies immunostained for ICAM-1 expression after topical capsaicin application to release cutaneous neuropeptides. Arrows, dermal microvascular structures. Human skin was left untreated (a) or treated with capsaicin cream (0.075%) and biopsied at 6 h (b), 24 h (c), or 48 h (d).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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-alpha 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 beta 2- and beta 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-gamma , TNF-alpha , 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-alpha -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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Ansel, J. C., C. A. Armstrong, I. S. Song, K. L. Quinlan, J. E. Olerud, S. W. Caughman, and N. W. Bunnett. Interactions of the skin and nervous system. J. Investig. Dermatol. Symp. Proc. 2: 23-26, 1997[Medline].

2.   Ansel, J., J. Brown, D. Payan, and M. Brown. Substance P selectively activates TNF-alpha gene expression in murine mast cells. J. Immunol. 150: 1-8, 1993[Abstract/Free Full Text].

3.   Ansel, J. C., A. H. Kaynard, C. A. Armstrong, J. Olerud, N. Bunnett, and D. Payan. Skin-nervous system interactions. J. Invest. Dermatol. 106: 198-204, 1996[Medline].

4.   Brown, J., P. Perry, S. Hefeneider, and J. Ansel. Neuropeptide modulation of keratinocyte cytokine production. In: Molecular and Cellular Biology of Cytokines, edited by J. J. Oppenheim, M. C. Powanda, M. J. Kluger, and C. A. Dinarello. New York: Wiley-Liss, 1990, p. 451-456.

5.   Buell, G., M. F. Schulz, S. J. Arkinstall, K. Maury, M. Missotten, N. Adami, F. Talabot, and E. Kawashima. Molecular characterisation, expression and localisation of human neurokinin-3 receptor. FEBS Lett. 299: 90-95, 1992[Medline].

6.   Caughman, S. W. Adhesion molecules: their roles in cutaneous biology and inflammation. Prog. Dermatol. 25: 1-8, 1991.

7.   Cornelius, L. A., N. Sepp, L.-J. Li, K. Degitz, R. A. Swerlick, T. J. Lawley, and S. W. Caughman. Selective upregulation of intercellular adhesion molecule (ICAM-1) by ultraviolet B in human dermal microvascular endothelial cells. J. Invest. Dermatol. 103: 23-28, 1994[Abstract].

8.   Degitz, K., L.-J. Li, and S. W. Caughman. Cloning and characterization of the 5'-transcriptional regulatory region of the human intercellular adhesion molecule 1 gene. J. Biol. Chem. 266: 14024-14030, 1991[Abstract/Free Full Text].

9.   Duff, J. L., K. L. Quinlan, L. L. L. Paxton, S. M. Niak, and S. W. Caughman. Pervanadate mimics INFgamma -mediated induction of ICAM-1 expression via activation of STAT proteins. J. Invest. Dermatol. 108: 295-301, 1997[Abstract].

10.   Dustin, M., and T. A. Springer. Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells. J. Cell Biol. 107: 321-331, 1988[Abstract].

11.   Erjavec, F., F. Lembeck, and T. Florjanc-Irman. Release of histamine by substance P. Naunyn Schmiedebergs Arch. Pharmacol. 317: 67-70, 1981[Medline].

12.   Fong, T. M., S. A. Anderson, H. Yu, R.-R. C. Huang, and C. D. Strader. Differential activation of intracellular effector by two isoforms of human neurokinin-1 receptor. Mol. Pharmacol. 41: 24-30, 1991[Abstract].

13.   Goebeler, M., U. Henseleit, J. Roth, and C. Sorg. Substance P and calcitonin gene-related peptide modulate leukocyte infiltration to mouse skin during allergic contact dermatitis. Arch. Dermatol. Res. 286: 341-346, 1994[Medline].

14.   Grady, E. F., P. Baluk, S. Bohm, P. D. Gamp, H. Wong, D. G. Payan, J. Ansel, A. L. Portbury, J. B. Furness, D. M. McDonald, and N. W. Bunnett. Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastrointestinal tract. J. Neurosci. 16: 6975-6986, 1996[Abstract/Free Full Text].

15.   Graham, A., B. Hopkins, S. J. Powell, P. Danks, and I. Briggs. Isolation and characterisation of the human lung NK-2 receptor gene using rapid amplification of cDNA ends. Biochem. Biophys. Res. Commun. 177: 8-16, 1991[Medline].

16.   Hartmeyer, M., T. Scholzen, E. Becher, R. S. Bhardwaj, T. Schwarz, and T. A. Luger. Human dermal microvascular endothelial cells express the melanocortin receptor type I and produce increased levels of IL-8 upon stimulation with a-melanocyte-stimulating hormone. J. Immunol. 159: 1930-1937, 1997[Abstract].

17.   Hartung, H., and K. Toyka. Activation of macrophages by substance P: induction of oxidative burst and thromboxane release. Eur. J. Pharmacol. 89: 301-305, 1983[Medline].

18.   Hershey, A., L. Plenzani, R. Woodward, R. Miledi, and J. Krause. Molecular and genetic characterization, functional expression, and mRNA expression patterns of a rat substance P receptor. Ann. NY Acad. Sci. 632: 63-78, 1991[Medline].

19.   Hogg, N. Roll, roll, roll your leukocyte gently down the vein. Immunol. Today 13: 113-115, 1992[Medline].

20.   Holzer, P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides. Neuroscience 24: 739-768, 1988[Medline].

21.   Hosoi, J., G. Murphy, C. Egan, E. Lerner, S. Grabbe, A. Asahina, and R. Granstein. Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363: 159-163, 1993[Medline].

22.   Hsu, S., L. Raine, and H. Fanger. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29: 577-580, 1981[Abstract].

23.   Kimball, E. S., F. J. Persico, and J. L. Vaught. Substance P, neurokinin A, and neurokinin B induce generation of IL-1 like activity in P388D1 cells. J. Immunol. 141: 3564-3569, 1988[Abstract/Free Full Text].

24.   Kramp, J., J. Brown, P. Cook, B. Russell, T. Lawley, C. Armstrong, and J. Ansel. Neuropeptide induction of human microvascular endothelial cell interleukin 8 (Abstract). J. Invest. Dermatol. 104: 586A, 1995.

25.  Krause, J. E., Y. Takeda, and A. D. Hershey. Structure, functions, and mechanisms of substance P receptor action. J. Invest. Dermatol. 98, Suppl. 6: 2S-7S, 1992.

26.   Kubota, Y., H. K. Kleinman, G. R. Martin, and T. J. Lawley. Role of laminin and basement membrane in the morphological differentiation of human dermal microvascular endothelial cells into capillary-like structures. J. Cell Biol. 107: 1589-1598, 1988[Abstract].

27.   Lawrence, M. B., and T. A. Springer. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859-873, 1991[Medline].

28.   Lotz, M., J. H. Vaughan, and D. A. Carson. Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science 241: 1218-1221, 1988[Medline].

29.   Matis, W. L., R. M. Lavker, and G. F. Murphy. Substance P induces the expression of an endothelial-leukocyte adhesion molecule by microvascular endothelium. J. Invest. Dermatol. 94: 492-495, 1990[Abstract].

30.   McGillis, J., M. Mitsuhashi, and D. Payan. Immunologic properties of substance P. In: Psychoneuroimmunology, edited by R. Ader, D. Felten, and N. Cohen. San Diego, CA: Academic, 1991, p. 209-231.

31.   Nagy, J., L. Iversen, M. Goedert, D. Chapman, and S. Hunt. Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat. J. Neurosci. 3: 399-406, 1983[Abstract].

32.   Naik, S. M., N. Shibagaki, L.-J. Li, K. L. Quinlan, L. L. L. Paxton, and S. W. Caughman. Interferon gamma -dependent induction of human intercellular adhesion molecule-1 gene expression involves activation of a distinct STAT protein complex. J. Biol. Chem. 272: 1283-1290, 1997[Abstract/Free Full Text].

33.   Nakagawa, N., I. Iwamoto, and S. Yoshida. Effect of substance P on the expression of an adhesion molecule ICAM-1 in human vascular endothelial cells. Regul. Pept. 46: 223-224, 1993[Medline].

34.   Okamoto, A., M. Lovett, D. G. Payan, and N. W. Bunnett. Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK-1) receptor expressed in mammalian cells. Biochem. J. 299: 683-693, 1994[Medline].

35.   Paxton, L. L. L., L.-J. Li, V. Secor, J. L. Duff, S. M. Naik, N. Shibagaki, and S. W. Caughman. Flanking sequences for the human intercellular adhesion molecule-1 NF-kappa B response element are necessary for tumor necrosis factor alpha -induced gene expression. J. Biol. Chem. 272: 15928-15935, 1997[Abstract/Free Full Text].

36.   Payan, D., D. Brewster, and E. Goetzl. Specific stimulation of human T lymphocytes by substance P. J. Immunol. 131: 1613-1615, 1983[Abstract/Free Full Text].

37.   Payan, D., J. Levine, and E. Goetzl. Modulation of immunity and hypersensitivity by sensory neuropeptides. J. Immunol. 132: 1601-1604, 1984[Free Full Text].

38.   Pober, J. S., P. Bevilacqua, D. L. Mendrick, L. A. Lapierre, W. Fiers, and M. A. Gimbrone. Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol. 136: 1680-1687, 1985[Abstract/Free Full Text].

39.   Roe, M. W., J. J. Lemasters, and B. Herman. Assessment of fura-2 for measurements of cytosolic free calcium. Cell Calcium 11: 63-73, 1990[Medline].

40.   Rothlein, R., M. L. Dustin, S. D. Marlin, and T. A. Springer. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J. Immunol. 137: 1270-1274, 1986[Abstract/Free Full Text].

41.   Saban, M. R., R. Saban, D. Bjorling, and M. Haak-Frendscho. Involvement of leukotrienes, TNF-alpha , and the LFA-1/ICAM-1 interaction in substance P-induced granulocyte infiltration. J. Leukoc. Biol. 61: 445-451, 1997[Abstract].

42.   Sepp, N. T., J. Gille, L.-J. Li, S. W. Caughman, T. J. Lawley, and R. A. Swerlick. A factor in human plasma permits persistent expression of E-selectin by human endothelial cells. J. Invest. Dermatol. 102: 445-450, 1994[Abstract].

43.   Smith, C. H., J. N. W. N. Barker, R. W. Morris, D. M. MacDonald, and T. H. Lee. Neuropeptides induce rapid expression of endothelial cell adhesion molecules and elicit granulocytic infiltration in human skin. J. Immunol. 151: 3274-3282, 1993[Abstract/Free Full Text].

44.   Staunton, D. E., S. D. Marlin, M. L. Stratowa, M. L. Dustin, and T. A. Springer. Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 52: 925-933, 1988[Medline].

45.   Swerlick, R. A., E. Garcia-Gonzolez, Y. Kubota, Y. Xu, and T. J. Lawley. Studies of the modulation of MHC antigen and cell adhesion molecule expression on human dermal microvascular endothelial cells. J. Invest. Dermatol. 97: 190-196, 1991[Abstract].

46.   Swerlick, R. A., and T. J. Lawley. Role of microvascular endothelial cells in inflammation. J. Invest. Dermatol. 100: 111S-115S, 1993[Abstract].

47.   Vigna, S. R., J. J. Bowden, D. M. McDonald, J. Fisher, A. Okamoto, D. C. McVey, D. G. Payan, and N. W. Bunnett. Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells. J. Neurosci. 14: 834-845, 1994[Abstract].

48.   Von Andrian, U. H., J. D. Chambers, L. M. McEvoy, R. F. Bargatze, K.-E. Arfors, and E. C. Butcher. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. Proc. Natl. Acad. Sci. USA 88: 7538-7542, 1991[Abstract].

49.   Wang, L., M. Hilliges, T. Jernberg, D. Wiegleb-Edstrom, and O. Johansson. Protein gene product 9.5-immunoreactive nerve fibres and cells in human skin. Cell Tissue Res. 261: 25-33, 1990[Medline].

50.   Weisner-Menzel, L., B. Schultz, F. Vakilzadeh, and B. M. Czarnetzki. Electron microscopic evidence for a direct contact between nerve fibres and mast cells. Acta Derm. Venereol. 61: 465-469, 1981[Medline].

51.   Wiederman, C., F. Wiederman, A. Apperl, G. Kieselbach, G. Konwalinka, and H. Braunsteiner. In vitro human polymorphonuclear leukocyte chemokinesis and human monocyte chemotaxis are different activities of aminoterminal and carboxyterminal substance P. Naunyn Schmiedebergs Arch. Pharmacol. 340: 185-190, 1989[Medline].


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