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The Journal of Immunology, 2001, 166: 617-623.
Copyright © 2001 by The American Association of Immunologists

Necessity of Thromboxane A2 for Initiation of Platelet-Mediated Contact Sensitivity: Dual Activation of Platelets and Vascular Endothelial Cells1

Motoki Mitsuhashi*, Akane Tanaka*, Chie Fujisawa*, Keiko Kawamoto*, Atsuko Itakura*, Mikio Takaku{dagger}, Takasi Hironaka, Shuzo Sawada and Hiroshi Matsuda2,*

* Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan; {dagger} Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; and àPreclinical Research, Bayer Yakuhin, Ltd., Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the crucial role of platelet-derived thromboxane A2 (TXA2) in initiating Ag-specific contact sensitivity (CS), a platelet-dependent CS model using genetically mast cell-deficient W/Wv mice, was provided. In vivo treatment with BAYu3405, a TXA2 receptor antagonist, markedly suppressed CS responses in a dose-dependent manner. This inhibitory effect occurred when BAYu3405 was administered before an early initiating phase, suggesting that TXA2 may be a potent initiator of platelet-mediated CS responses. When platelets were pretreated with BAYu3405 in vitro, platelet aggregation as well as serotonin release, which is able to induce the early phase response allowing local recruitment of CS effector T cells due to direct activation of vascular endothelial cells, was inhibited. The addition of U46619, a TXA2 agonist, or a mixture of platelets and thrombin-enhanced expression of both ICAM-1 and VCAM-1 on isolated mouse aortic endothelial cells, which was completely abolished by pretreatment with BAYu3405. Furthermore, intradermal injection of U46619 into the ear of platelet-depleted mice led to CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. These findings suggest that TXA2 generated from platelets activated with Ag may mediate initiation of CS responses through inducing serotonin release from platelets and the subsequent aggregation and up-regulated expression of ICAM-1 and VCAM-1 on vascular endothelial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Platelets possess a broad range of biologically active materials capable of inducing a chain reaction for hemostasis, coagulation, and thrombosis (1). In addition, these materials are strongly involved in the primary response of allergic and nonallergic inflammation (2). Following immunological or nonimmunological activation, platelets rapidly release mediators stored in dense or {alpha} granules such as ADP, ATP, serotonin, von Willebrand factor (vWF),3 various kinds of cytokines and chemokines, and newly generated mediators derived from membrane phospholipids (2). Serotonin directly activates endothelial cells of the local vascular capillaries, inducing an increase in permeability that characterizes early phase responses of cutaneous contact sensitivity (CS), and allows local recruitment into the tissues of CS effector T cells to mediate the late phase responses of CS (3). Although mast cells are the major source of skin serotonin in rodents, serotonin stored in platelets also appears to act as a critical mediator to initiate CS responses. In fact, genetically mast cell-deficient W/Wv and Sl/Sld mice manifest relatively intact CS responses, which are abolished by depletion of circulating platelets or by in vivo pretreatment with a 5-HT2A serotonin receptor antagonist (4, 5). Recently, we have demonstrated that transfer of human platelets sensitized with IgE Ab analogous to CS-initiating factors is able to initiate Ag-specific T cell-dependent CS responses through local serotonin release in mast cell-deficient mice and their normal littermates (6).

Thromboxane A2 (TXA2), another potent vasoconstrictor (7, 8, 9), is generated by activation of platelet membrane phospholipase A2 following adhesion of platelets to collagen and the basement membrane of exposed subendothelium in the presence of vWF and platelet glycoprotein Ib (10). Furthermore, rapidly released TXA2 leads to the recruitment of circulating platelets into the affected site and to the aggregation of platelets through binding to endoperoxide/TXA2 receptors collaborating with ADP (11). The fact that cross-linking of the Fc{epsilon}RI with Ag induces an immediate increase in cytosolic phospholipase A2 in mouse cultured mast cells (12) and, furthermore, that human and mouse platelets constitutively express functional Fc{epsilon}RI (6, 13, 14) suggests that platelets may be capable of releasing TXA2 following Ag-specific activation through IgE Ab or CS-initiating factors. Furthermore, recent studies have shown that human endothelial cells express TXA2 receptors (15), and that U-46619, a stable TXA2 analog, induces significant expression of adhesion molecules on the surface of human vascular endothelial cells such as ICAM-1 and VCAM-1 (16). These findings give rise to a possibility that, in the process of CS responses, TXA2 released from platelets may lead to the expression of such adhesion molecules on the vascular endothelial cells at the affected site, contributing to local recruitment of CS effector T cells. In this study, we assessed the possible involvement of TXA2 released from platelets in eliciting platelet-mediated CS responses in mast cell-deficient W/Wv mice by using a specific antagonist to the TXA2 receptor. We demonstrated that platelet TXA2 was essential for initiation of CS early phase responses because it induced subsequent serotonin release, platelet aggregation, and expression of ICAM-1 and VCAM-1 on vascular endothelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male genetically mast cell-deficient WBB6F1-W/Wv mice and their control mast cell-sufficient WBB6F1+/+ mice (7–10 wk of age) were purchased from Japan SLC (Hamamatsu, Japan), and mice were kept in our laboratory for at least 1 wk before use.

Ab and other reagents

Hamster anti-mouse CD54 (ICAM-1) mAb and rat anti-mouse CD106 (VCAM-1) mAb were purchased from PharMingen (San Diego, CA), and sheep anti-rat vWF polyclonal Ab was obtained from Cedarlane Laboratories (Hornby, Ontario, Canada). Peroxidase-conjugated goat anti-rat IgG polyclonal Ab, peroxidase-conjugated goat anti-hamster IgG polyclonal Ab, and FITC-conjugated donkey anti-sheep IgG polyclonal Ab were provided by Jackson ImmunoResearch (West Grove, PA). BAYu3405, obtained from Bayer Yakuhin (Osaka, Japan), was dissolved at a concentration of 10-2 M in DMSO, and serial dilutions were prepared in PBS. All chemicals used were purchased from Sigma (St. Louis, MO) unless otherwise indicated.

CS responses

Mast cell-deficient WBB6F1-W/Wv mice were sensitized and challenged with picryl chloride (PCl; Tokyo Kasei Industries, Tokyo, Japan) according to the method reported previously (5). Briefly, 150 µl of 5% PCl dissolved in a mixture of ethanol and acetone (4:1) was applied to the clipped chest and abdomen and to the foot pads twice 4 and 7 days before challenge. Four days after the second sensitization, the mice were challenged by painting both sides of both ears with 1 drop of 0.8% purified PCl in olive oil. Ear thickness was measured with an engineer’s micrometer (Mitsutoyo, Tokyo, Japan) before the challenge and at 2 and 24 h after the challenge. The increment in ear thickness was expressed as units of 10-3 cm.

Preparation of anti-mouse platelet serum

Rabbit anti-mouse platelet serum was produced as described previously (17). To evaluate a platelet-depleting ability of serum samples, 200 µl of PBS-diluted anti-platelet serum (75 µl) was injected i.v. into mice, and blood cells collected from the retro-orbital plexus were counted at several time points with an automatic cell counter (Celltac {alpha} MEK-6158; Nihon Koden, Tokyo, Japan). Normal sera prepared from rabbits before the first immunization were used as a control.

In vivo treatment with anti-platelet serum, BAYu3405, or U46619

WBB6F1-W/Wv mice were challenged with PCl at 5 h after i.v. injection with anti-platelet or normal serum, or at several hours after i.p. injection with various doses of BAYu3405, a TXA2 receptor antagonist (18, 19, 20), or diluent alone. In other experiments, 50 µl of 10-5 M U46619, a TXA2 agonist (21), or diluent alone was injected intradermally into both ears of mice immediately before the challenge.

Measurement of tissue eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activities

To quantitatively assess inflammatory cells infiltrated into ears, we estimated tissue levels of EPO and MPO reflecting the presence of eosinophils and neutrophils, respectively, according to the methods reported (22, 23). Right ears were cut off at 24 h after the challenge, and round skin samples were enucleated from the proximal area using disposable skin punch equipment (5 mm in diameter). After homogenizing in 2 ml of 50 mM potassium phosphate containing 0.5% hexadecyl-trimethylammonium bromide, the specimens were centrifuged at 12,000 rpm for 10 min at room temperature, and supernatants were collected. EPO and MPO activities in the samples were measured by a microplate reader (ImmunoMini NJ-2300; Nalge Nunc International, Tokyo, Japan).

Histological examination

Left ears were removed at 24 h after the challenge and fixed in 10% phosphate-buffered formalin (pH 7.2). Paraffin sections (5-µm thick) were made by the routine method and stained with toluidine blue (pH 4.0) for counting mast cells (24), Congo red for counting eosinophils (25), and hematoxylin and eosin for counting mononuclear cells.

Immunohistochemistry

Small pieces were cut from ears of mice, embedded in OCT compound (Miles, Elkhard, IN), and snap frozen in liquid nitrogen. Frozen sections cut into 6-µm pieces were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 5 min on ice and washed with PBS twice. After treatment with PBS containing 0.1% NaN3 and 0.3% H2O2 to inhibit endogenous peroxidase, the sections were placed in 10% normal goat serum to block nonspecific protein binding. Hamster anti-mouse ICAM-1 mAb and rat anti-mouse VCAM-1 mAb (2.5 µg/ml in PBS with 1% BSA) were applied to the section samples and incubated overnight at 4°C. After washing twice, preparations were incubated with a 1:200 dilution of peroxidase-conjugated goat anti-rat IgG Ab for 30 min at room temperature. Visualization of the reaction products was performed with 0.2 mg/ml 3,3'-diaminobenzidine tetrahydrochloride in PBS supplemented with 0.003% H2O2.

Preparation of platelets

Human peripheral blood anticoagulated with 0.38% trisodium citrate was collected from volunteers who had no history of allergic diseases. Platelet-rich plasma (PRP) was obtained by centrifugation of blood samples for 13 min at 120 x g. Mouse citrated blood collected by cardiac puncture was diluted in modified Tyrode’s buffer (138 mM NaCl, 3 mM KCl, 12 mM NaHCO3, 1 mM MgCl2, 5 mM glucose, 0.4 mM Na2HPO4, and 10 mM HEPES) containing 1% BSA and 5 ng/ml PGI2, and PRP was obtained by centrifugation for 13 min at 120 x g. To prevent platelet activation, a high dose of PGI2 (300 ng/ml) was added to PRP (26). Platelets were washed three times with Tyrode’s buffer containing 1% BSA and 300 ng/ml PGI2 for 15 min at 1000 x g and resuspended in Tyrode’s buffer. Examination of the platelet preparations by Giemsa staining showed little contamination of other blood cell components.

Measurement of platelet aggregation and ATP release

Human PRP (400 µl) was incubated for 1 min at 37°C in glass cuvettes fixed in a lumi-aggregometer (C-500; Chrono-Log, Havertown, PA), which is capable of simultaneously recording platelet aggregation and release of ATP contained in platelet-dense granules (27). Fifty microliters of Chronolium (Chrono-Log) containing luciferin and luciferase was added to PRP, and followed by the addition of 50 µl of BAYu3405 diluted in Tyrode’s buffer. One minute later, thrombin or collagen was added at a final concentration of 0.1 U/ml or 0.5 µg/ml, respectively. Aggregation and ATP release were recorded for 10 min after the addition of the stimulants.

[3H]Serotonin release from platelets

Serotonin release from mouse platelets was measured according to the method described previously (6). Briefly, fresh murine platelets from PRP were incubated with 0.2 µCi/ml [3H]serotonin (Amersham Pharmacia Biotech, Tokyo, Japan) and 5 ng/ml PGI2 for 45 min at 25°C. After washing twice with Tyrode’s solution containing PGI2 (300 ng/ml), 108 platelets were resuspended in 1 ml of Tyrode’s solution with various concentrations of BAYu3405. Next, 108 platelets were incubated in 1.5-ml microtubes with 0.2 U/ml thrombin for 15 min at 37°C. One hundred microliters of platelet suspension was collected and then added to 100 µl of a stop solution (10 mM EDTA and 200 mM formaldehyde). After incubation for 30 min on ice, the tubes were centrifuged at 14,000 rpm for 2 min. Aliquots (100 µl) of the supernatants and the nonseparated suspensions were counted for {beta} emissions in a scintillation counter. [3H]Serotonin percent release was calculated as supernatant cpm/total cpm x 100. Results were expressed as net percent release (percent release of experimental groups minus percent release of background from platelets that were incubated with medium alone). Percent release of background tubes usually averaged ~5–10%.

Preparation of mouse aortic endothelial cells (AEC)

Mouse AEC were isolated by using a modification of the technique described previously (28). Under a stereoscopic microscope, a 24-gauge cannula was inserted at the proximal portion of the thoracic aorta, and the abdominal aorta was cut at the middle portion. The distal end of the fragment was fastened with a nylon thread, and then FCS-free DMEM containing 0.2% collagenase type 2 was gently poured from the proximal end of the fragment. After incubation for 30 min at 37°C, AEC were removed from the aorta by flushing with 5 ml of DMEM with 10% FCS. Each cell cluster picked up by using a micropipette was put into each well of 24-well culture plates (Nage Nunc International, Roskilde, Denmark) containing DMEM supplemented with 20% FCS, 2 mM L-glutamine, 25 mM HEPES, 50 U/ml penicillin, and 50 µg/ml streptomycin, and allowed to culture for ~1–2 wk at 37°C in a humidified atmosphere flushed with 5% CO2 in air. The culture medium was replaced with 1 ml of fresh culture medium every week. Before reaching 80% confluence, the cells were passed in 60-mm culture dishes by a trypsin and EDTA solution. When the cells reached 80% confluence, they were harvested and resuspended at a concentration of 2 x 105 cells/ml in culture medium. A total of 4 x 104 cells were seeded in each well of 96-well culture plates and eight-well chamber slides (Nage Nunc International) that were coated with Cellmatrix Type 1-A (Nitta Gelatin, Osaka, Japan) according to the manufacturer’s instructions, and cultured for 1 wk at 37°C in a humidified atmosphere flushed with 5% CO2 in air. The cells cultured in eight-well chamber slides were checked by morphology, uptake of acetylated low density lipoprotein (Ac-LDL), and expression of vWF (29).

Detection of adhesion molecules expressed on AEC

AEC cultured for 1 wk in Cellmatrix Type 1-A-coated 96-well microplates were allowed to examine expression of ICAM-1 and VCAM-1. AEC were preincubated with various concentrations of BAYu3405 for 5 min and then incubated with 10-5 M U46619 for 20 h (ICAM-1) and for 12 h (VCAM-1) at 37°C in a humidified atmosphere flushed with 5% CO2 in air. After incubation with the stimulants and/or BAYu3405, AEC were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min on ice and washed with PBS three times. Each well was treated with blocking buffer for 1 h, and AEC were incubated with 50 µl of PBS containing 1% BSA and 1 µg/ml hamster anti-mouse ICAM-1 mAb or 5 µg/ml rat anti-mouse VCAM-1 mAb for 1 h at room temperature. After washing with PBS three times, AEC were reincubated with a 1:20,000 dilution of peroxidase-conjugated goat anti-rat IgG polyclonal Ab or peroxidase-conjugated goat anti-hamster IgG polyclonal Ab for 1 h. The reaction products were visualized by incubation with 0.2 mg/ml 3,3'-diaminobenzidine tetrahydrochloride in PBS supplemented with 0.003% H2O2 for 5 min, and the absorbance at 490 nm was measured by a microplate reader. In other experiments, AEC were incubated with various concentrations of BAYu3405 for 5 min, and then 3 x 106 mouse platelets and 1 U/ml thrombin were added.

Statistical analysis

A two-tailed Student’s t test was performed for statistical analysis of the data, and p < 0.05 was taken as the level of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduction in CS responses in W/Wv mice injected with anti-platelet serum

To examine the role of platelets in CS responses, Ab against mouse platelets were injected into mast cell-deficient W/Wv mice before challenge with PCl. First, in the preliminary experiment, single i.v. injection of anti-platelet serum rapidly induced drastic reduction in the number of circulating platelets (<10%) within 5 h, and this effect lasted for >24 h. Therefore, we administered a single injection of anti-platelet serum at 5 h before the challenge. Although W/Wv mice injected with control serum manifested late phase ear swelling responses without a detectable macroscopic early phase response at 2 h, the injection with anti-platelet serum decreased elicitation of the late phase ear swelling responses (Fig. 1GoA). The ears of W/Wv mice injected with control serum or anti-platelet serum were collected and processed for EPO and MPO assays after the final measurement of ear thickness. The injection of anti-platelet serum led to a significant decrease in levels of both EPO and MPO as compared with mice injected with control serum (Fig. 1GoB). Thus, CS responses were dependent on platelets in mast cell-deficient mice.



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  		FIGURE 1. Effect of platelet depletion on PCl-induced CS responses in mast cell-deficient W/Wv mice. W/Wv mice were injected with anti-platelet serum (filled columns) or normal serum (open columns) at 5 h before challenge with PCl. Ear swelling was determined at 2 and 24 h after the challenge (A), and tissue EPO and MPO activities were measured at 24 h after the challenge (B). Each point represents the mean ± SE of four separate experiments. *, p < 0.001, when compared with normal serum.

 
Reduction in CS responses in W/Wv mice injected with a TXA2 receptor antagonist

Platelets release various kinds of chemical mediators stored in cytoplasmic granules and generated newly after stimulation. Therefore, we investigated whether TXA2 influenced the elicitation of CS induced by active sensitization using BAYu3405, a selective TXA2 receptor antagonist. Thus, single i.p. injection of various doses of BAYu3405 was performed at 1 h before challenge of W/Wv mice with PCl. Fig. 2Go shows that the injection of BAYu3405 significantly decreased elicitation of late ear swelling responses in a dose-dependent manner; 250 mg/kg body weight led to the maximum inhibitory effect, which was comparable to nonimmunized control W/Wv mice. Next, histological examination was performed on the ears at 24 h after the challenge. No or very few mast cells were detected in the ears of all groups of mice. Active sensitization with PCl induced marked infiltration of eosinophils, neutrophils, macrophages, and lymphoid cells in mice injected with diluent alone, but not in mice injected with 250 mg/kg body weight of BAYu3405.



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  		FIGURE 2. Inhibitory effect of a TXA2 receptor antagonist BAYu3405 on CS responses. W/Wv mice were injected i.p. with various doses of BAYu3405 at 1 h before the challenge with PCl. Each point represents the mean ± SE of four separate experiments. *, p < 0.05, when compared with diluent instead of BAYu3405.

 
CS initiation suppressed by a TXA2 receptor antagonist

Our previous study (6) suggested that platelet-mediated CS responses consist of two distinct components: 1) an early initiating phase, which was optimal at 2 h after the challenge; and 2) a late effector phase, which was optimal 24 h later. To determine the phase point where TXA2 affected the process of CS elicitation, W/Wv mice were injected with 50 mg/kg body weight of BAYu3405 at various time points before or after the PCl challenge. When the injection of BAYu3405 was performed at 2 h after the challenge, the inhibitory ability of BAYu3405 on ear swelling responses was completely suppressed (Fig. 3Go). Thus, a TXA2 receptor antagonist was effective at the early phase of CS responses.



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  		FIGURE 3. Effect on CS responses of BAYu3405 given at various times before and after the challenge with PCl. Immunized and nonimmunized W/Wv mice were injected i.p. with 50 mg/kg BAYu3405 or diluent alone, and ear swelling was determined at 24 h after the challenge. Each point represents the mean ± SE of four separate experiments. *, p < 0.001, when compared with immunized mice treated with diluent alone.

 
Platelet aggregation and ATP release suppressed by a TXA2 receptor antagonist

Platelets generate and release TXA2 immediately following stimulation, and the TXA2 is capable of inducing a subsequent activation of platelets, which leads to drastic morphological changes and the release of a variety of chemical mediators. Experiments were conducted to determine the platelet-activating effects of TXA2 released from platelets. When human platelets were stimulated with optimal doses of thrombin and collagen, rapid aggregation of platelets and ATP release from platelets were observed; the pretreatment with various doses of BAYu3405 inhibited these phenomena in a dose-dependent manner (Fig. 4Go).



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  		FIGURE 4. Inhibitory effects of BAYu3405 on platelet aggregation and ATP release from platelets. Human PRP was incubated with various doses of BAYu3405 before the addition of 0.1 U/ml thrombin (A and C) or 0.5 µg/ml collagen (B and D). Aggregation (A and B) and ATP release (C and D) were measured by a lumi-aggregometer.

 
Serotonin release from platelets inhibited by a TXA2 receptor antagonist

Serotonin released from platelets following local Ag challenge is essential for platelet-mediated CS initiation due to activation of vascular endothelial cells and subsequent recruitment and activation of CS effector T cells through their surface serotonin receptor (6, 30). Therefore, we next investigated whether BAYu3405 was capable of suppressing serotonin release from platelets stimulated with 0.2 U/ml thrombin. As shown in Fig. 5Go, treatment of mouse platelets with thrombin induced a marked release of serotonin. Pretreatment with BAYu3405 led to significant suppression of serotonin release from platelets in a dose-dependent manner; a dose of 10-5 M maximally reduced serotonin release by 64%, as compared with pretreatment with diluent alone (Fig. 5Go).



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  		FIGURE 5. Inhibitory effects of BAYu3405 on [3H]serotonin release from murine platelets stimulated with thrombin. A total of 108 murine platelets were incubated with 0.2 U/ml thrombin for 15 min at 37°C after pretreatment with various concentrations of BAYu3405. Each point represents the mean ± SE of four separate experiments using duplicate samples. *, p < 0.005, when compared with platelets pretreated with diluent alone.

 
Expression of VCAM-1 on peripheral blood vessels in the ear

Vascular endothelial cells constitutively express TXA2 receptors, and the binding of TXA2 leads to the expression of adhesion molecules on their surface (15, 16). VCAM-1 is an adhesion molecule expressed mainly on vascular endothelial cells following local stimulation and one that is responsible for extravasation of T cells (31). Given that elicitation of CS late phase responses is provoked by recruitment of effector T cells at the site of Ag challenge (3), immunohistochemical analysis for VCAM-1 was provided in the ears of actively sensitized W/Wv mice injected with diluent alone or 50 mg/kg body weight of BAYu3405. Fig. 6Go clearly shows that a strongly positive reaction for VCAM-1 was detected in the vascular endothelium of the ears in W/Wv mice injected with diluent alone. In contrast, in W/Wv mice injected with BAYu3405 no or very weak reaction was observed, which was comparable to that in nonimmunized control mice.



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  		FIGURE 6. Immunohistochemical analysis for VCAM-1 in the ear at 24 h after the challenge with PCl in W/Wv mice. Vascular endothelium shows positive reaction for protein of VCAM-1 in the ear section of immunized mice injected with diluent alone (B). In contrast, no or little positive reaction is observed in the sections of immunized mice injected with BAYu3405 (C) or of nonimmunized mice injected with diluent alone (A). Original magnification, x160.

 
Direct effect of TXA2 on expression of ICAM-1 and VCAM-1 on mouse AEC

The above findings suggested the involvement of TXA2 in VCAM-1 expression at affected tissue sites. To further clarify the direct effect of TXA2, primary cultures of mouse AEC were prepared. Cells isolated from the thoracic artery displayed endothelial cell characteristics as demonstrated by a monolayer of the cells showing cobblestone formation, uptake of Ac-LDL, and expression of vWF (Fig. 7Go). AEC pretreated with 10-5 M BAYu3405 or its diluent were incubated for 20 h (ICAM-1) or for 12 h (VCAM-1) in the presence of 10-5 M U46619, a TXA2 agonist. Treatment with U46619 increased expression of both ICAM-1 and VCAM-1, which was completely blocked by pretreatment with BAYu3405 (Fig. 8Go).



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  		FIGURE 7. Uptake of Ac-LDL and detection of vWF in isolated mouse AEC. Cells isolated from the abdominal aorta were assessed for identification of endothelial cells as described in Materials and Methods. Cells show positive reaction for Ac-LDL uptake (left) and vWF (right). Original magnification, x320.

 


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  		FIGURE 8. Inhibitory effect of BAYu3405 on ICAM-1 and VCAM-1 expression on mouse AEC stimulated with a TXA2 agonist. AEC were incubated with 10-5 M U46619 after preincubation with 10-5 M BAYu3405 for 5 min. An ELISA for ICAM-1 and VCAM-1 was performed as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples. *, p < 0.05, when compared with cells treated with diluent alone. {dagger}, p < 0.05, when compared with cells pretreated with diluent and treated with U46619.

 
To investigate TXA2, AEC were incubated with mouse platelets stimulated with 1 U/ml thrombin. Expression of ICAM-1 and VCAM-1 was enhanced in the presence of platelets or thrombin alone, and incubation with both platelets and thrombin produced an additive effect (Fig. 9Go). When AEC were pretreated with various doses of BAYu3405, the expression of both ICAM-1 and VCAM-1 enhanced by the costimulation with platelets and thrombin was inhibited in a dose-dependent manner (Fig. 9Go).



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  		FIGURE 9. Inhibitory effect of BAYu3405 on ICAM-1 and VCAM-1 expression on mouse AEC stimulated with platelets and thrombin. AEC were incubated with 3 x 106 mouse platelets and 1 U/ml thrombin after preincubation with various concentrations of BAYu3405 for 5 min. An ELISA for ICAM-1 and VCAM-1 was performed as described in Materials and Methods. Each point represents the mean ± SE of three separate experiments using duplicate samples. *, p < 0.001, when compared with cells treated with platelets or thrombin alone. {dagger}, p < 0.05, when compared with cells pretreated with diluent and treated with platelets and thrombin.

 
CS initiation mediated by local injection of a TXA2 agonist

To investigate TXA2 further, U46619, a TXA2 agonist, was injected intradermally into the ears of platelet-depleted mice immediately before the challenge with PCl. When circulating platelets in immunized mice were depleted by injection with anti-platelet Ab, late ear swelling responses were abolished, and weak reaction for ICAM-1 (24 h later) and VCAM-1 (12 h later) was observed on the vascular endothelium in the ear (Table IGo). In contrast, local injection with U46619 led not only to significant ear swelling responses in the immunized and platelet-depleted mice but also to marked expression of both the adhesion molecules on the endothelium of many blood vessels, which was roughly comparable to the results for platelet-rich mice immunized with PCl (Table IGo).


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  		Table I. CS initiation and expression of adhesion molecules mediated by local injection of U46619, a TXA2 agonist

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adhesion and activation of circulating platelets at the vascular endothelium of local injured or inflamed tissues through an interaction with platelet glycoprotein Ib receptor complex or Fc{epsilon}RI are the primary integrated events in the process of the hemostatic response and the allergic and nonallergic responses (2). Activated platelets release a variety of biologically active mediators from dense and {alpha} granules and generate metabolic products of membrane arachidonic acid. TXA2 functions as a stimulator of platelet aggregation and a strong constrictor of vascular and bronchial smooth muscles through the TXA2 receptor, which is directly involved in the pathogenesis of many cardiovascular and allergic diseases such as stroke, bronchial asthma, and allergic rhinitis (32, 33, 34). In this study, platelet depletion in response to the injection with anti-platelet Ab abrogated elicitation of PCl-specific CS responses in mast cell-deficient W/Wv mice, which reconfirmed the result reported by Geba et al. (4). Here, we provided novel evidence that mast cell-independent CS initiation was largely blocked by in vivo treatment with BAYu3405, an antagonist of the TXA2 receptor. These findings strongly suggested the necessity of platelet TXA2 in platelet-mediated CS responses. Recently, we have demonstrated that i.v. injection of Ag-specific CS effector T cells that were depleted of CS-initiating cells together with human platelets presensitized in vitro with Ag-specific IgE mAb led to the elicitation of CS responses in recipient mice. Furthermore, the platelet-mediated CS responses were induced through serotonin locally released from platelets activated by topical application with Ag (6). In CS responses, serotonin is considered to directly activate vascular endothelial cells at the early phase and to lead local infiltration of CS effector T cells. When W/Wv mice were treated with BAYu3405 in vivo at various time points before or after the challenge with PCl, the inhibitory effect was obvious at only 1 h before and 1 h after the challenge, suggesting that TXA2 may primarily act at the early phase of CS responses rather than the late effector phase. In fact, pretreatment of human platelets with BAYu3405 in vitro resulted in marked, but not complete, suppression of platelet aggregation and serotonin release by collagen or thrombin. In the primary phase of hemostatic processes, ADP, serotonin, and TXA2 are rapidly released from platelets after platelet adhesion, and TXA2 promotes further release of strong agonists including serotonin from activated platelets, thereby contributing to thrombin formation (1, 2). Thus, we concluded that TXA2 released from platelets following the challenge with Ag may be essential for the induction of further release of serotonin and other minor CS mediators from platelets and the subsequent platelet aggregation at the affected site.

Tight adhesion of effector T cells to vascular endothelium is an important step in the transmigration of endothelium at the inflamed site. Activated T cells express both {beta}2 integrin LFA-1 and {beta}1 integrin very late antigen (VLA)-4, which bind to ICAM-1 and VCAM-1, respectively, on peripheral vascular endothelial cells (35). Previous studies demonstrated that both adhesion molecules were induced or increased on vascular endothelium at the CS sites (36, 37) and that CS elicitation was suppressed in mice treated with Ab to ICAM-1 and LFA-1 (38) and in mice treated with anti-{alpha}4 integrin mAb, which blocked adhesion to VCAM-1 (39). The additional finding that stimulation of human vascular endothelial cells with TXA2 enhanced expression of ICAM-1 and VCAM-1 (16) suggested a possible role of TXA2 in promoting the expression of adhesion molecules on vascular endothelial cells in the process of platelet-mediated CS responses. To clarify this point, the in vivo and in vitro experiments were conducted. In vivo treatment with BAYu3405 inhibited the expression of VCAM-1 on vascular endothelium in ears challenged with PCl, and intradermal injection of U46619, a TXA2 agonist, into the ear of platelet-depleted mice induced CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. Next, we attempted to examine the direct effect of TXA2 on the expression of adhesion molecules by using mouse AEC isolated according to our original technique. The addition of U46619 or mouse platelets and thrombin led to enhanced expression of both ICAM-1 and VCAM-1; this promoting effect was completely eliminated by pretreatment with BAYu3405. Thus, TXA2 derived from activated platelets may directly stimulate vascular endothelial cells through the TXA2 receptor and enhance expression of ICAM-1 and VCAM-1, thereby contributing to extravasation of CS effector T cells that mediate CS late phase responses. Both serotonin and TXA2 induce vasoconstriction (11), which may reduce blood stream velocity and assist platelet adhesion and the subsequent adhesion of CS effector T cells. Furthermore, because the addition of both serotonin and TXA2 to endothelial cells increased the number of each receptor and cell proliferation as compared with their individual effects (40), TXA2 and serotonin may synergistically activate vascular endothelial cells.

Many investigators reported that administration of a TXA2 antagonist was capable of preventing the pathophysiological parameters of bronchial asthma and allergic rhinitis in experimental animals and patients (41, 42, 43, 44). This study clearly demonstrated that in vivo treatment of mast cell-deficient mice with BAYu3405 led to reduction in platelet-mediated CS responses, thus making these mice a suitable animal model for human CS. Thus, we concluded that a TXA2 antagonist has the potential to act as a therapeutic agent for human CS.


   Acknowledgments
 
We thank Dr. T. Kodama (Department of Molecular Biology and Medicine, University of Tokyo, Tokyo, Japan) for technical support.


   Footnotes
 
1 This work was supported in part by grants from the Ministry of Education, Science Sports, and Culture, Japan and from the Specially Promoted Research on Atopic Disorders from the Tokyo Metropolitan Government. Back

2 Address correspondence and reprint requests to Dr. Hiroshi Matsuda, Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Sawai-cho, Fuchu, Tokyo 183-8509, Japan. Back

3 Abbreviations used in this paper: vWF, von Willebrand factor; Ac-LDL, acetylated low density lipoprotein; CS, contact sensitivity; AEC, aortic endothelial cells; EPO, eosinophil peroxidase; MPO, myeloperoxidase; PCl, picryl chloride; PRP, platelet-rich plasma; TXA2, thromboxane A2. Back

Received for publication April 5, 2000. Accepted for publication October 10, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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