Introduction

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A central step in the migration of circulating leukocytes from blood to extravascular tissue is their adhesion to vascular endothelium. The process of leukocyte binding and transmigration is complex and involves the sequential interaction of several specific adhesion molecules located on both leukocyte and endothelial cell surfaces (Bevilacqua, 1993; Carlos and Harlan, 1994). Members of the selectin family of adhesion molecules are implicated in the initial stages of leukocyte capture (Bevilacqua and Nelson, 1993). One member of this family, P-selectin, is a glycoprotein found in the alpha granules of platelets (Hsu-Lin et al., 1984; Stenberg et al., 1985) and also in cytoplasmic granules of endothelial cells known as Weibel-Palade bodies (Bonfanti et al., 1989; McEver et al., 1989). Exposure of endothelial cells to appropriate stimuli leads to the mobilization of P-selectin from its intracellular stores and expression of this ligand on the endothelial cell surface (Hattori et al., 1989a; McEver et al., 1989). To date, expression of P-selectin has been shown to be rapidly induced (within minutes) after exposure of endothelial cells to a variety of inflammatory stimuli including histamine (Hattori et al., 1989a; McEver et al., 1989; Lorant et al., 1991), thrombin (Hattori et al., 1989a; Lorant et al., 1991) and components of complement (Hattori et al., 1989b; Foreman et al., 1994).

The cysteinyl leukotrienes LTC₄, LTD₄ and LTE₄ are biologically active lipid mediators derived from the metabolism of arachidonic acid (Samuelsson, 1983). A substantial body of evidence now exists showing that these mediators are potent proinflammatory agents (Samuelsson et al., 1987; Henderson, 1994; Hay et al., 1995) and they have been implicated in the pathogenesis of inflammatory disease in a variety of tissues and organs (reviewed in Henderson, 1994; Hay et al., 1995; Drazen, 1995). Among the proinflammatory actions of these mediators are effects on the vasculature to cause increased vascular reactivity (Drazen et al., 1980) and permeability (Dahlén et al., 1981; Joris et al., 1987; Evans et al., 1989), as well as effects on inflammatory cell motility (Spada et al., 1994) and recruitment of leukocytes into tissue (Foster and Chan, 1991; Laitinen et al., 1993). Although the precise mechanism(s) by which cysteinyl leukotrienes cause leukocyte recruitment is not yet clear, an area of emerging interest is in the ability of these mediators to affect the function and/or surface expression of cell adhesion molecules. Several observations indicate that cysteinyl leukotrienes may be involved in leukocyte recruitment via actions at the level of the endothelial cell involving P-selectin. Incubation of cultured HUVEC with LTC₄ or LTD₄ has been shown to induce rapid surface expression of P-selectin (Datta et al., 1995; Papayianni et al., 1996) and increased adhesion of neutrophils to HUVEC monolayers (McIntyre et al., 1986; Papayianni et al., 1996) via a mechanism involving P-selectin (Papayianni et al., 1996). In addition, administration of LTC₄ in vivo produces an increase in the rolling flux of leukocytes in rat mesenteric microvessels through a mechanism also dependent on P-selectin (Kanwar et al., 1995).

Cysteinyl leukotrienes mediate their biological effects via interaction with specific receptors located in target cells. Evidence is accumulating that heterogeneity within the leukotriene receptor population exists. In tissues such as guinea pig airways and ileum, findings from functional (Fleisch et al., 1982; Snyder and Krell, 1984; Gardiner et al., 1990) and receptor binding (Pong and DeHaven, 1983; Hogaboom et al., 1983) studies indicate the presence of different types of cysteinyl leukotriene receptors, one which is stimulated by LTC₄ and a second which is activated by LTD₄/LTE₄ (Snyder and Krell, 1984; Gardiner et al., 1990). The existence of more than one cysteinyl leukotriene receptor has also been reported in both the ferret spleen (Gardiner et al., 1994) and sheep airways (Cuthbert et al., 1991; Gardiner et al., 1994). In contrast, in airway smooth muscle preparations from human lung it is thought that only a single type of cysteinyl leukotriene receptor exists (Buckner et al., 1986). This CysLT₁ receptor is characterized by contractions which are inhibited by potent CysLT₁ receptor antagonists (Buckner et al., 1986, 1990; Hay et al., 1987; Fujiwara et al., 1993) and it is thought that LTC₄ and LTD₄ are both active at this site (Gardiner et al., 1994). There is some evidence to support the existence of leukotriene receptor subtypes in human tissue. Based on the results from inhibition studies with leukotriene receptor antagonists, distinct receptors for cysteinyl leukotrienes have been identified in human bronchial smooth muscle and pulmonary vein (Labat et al., 1992).

Little is known regarding the possible receptor mechanism(s) involved in mediating the actions of cysteinyl leukotrienes to induce endothelial P-selectin expression. Given the potential importance of adhesion molecule expression in inflammation and inflammatory disease we were interested in investigating the effects of cysteinyl leukotrienes on P-selectin expression in human endothelial cells, particularly in terms of characterizing the receptor(s) involved. In the present study we have examined the ability of cysteinyl leukotrienes to induce P-selectin expression in cultured HUVEC and have assessed the effects of a number of CysLT₁ receptor antagonists on these responses. For comparison, the effects of histamine and of selective histamine receptor antagonists on histamine-induced endothelial P-selectin expression have also been reported. Our findings suggest that in human endothelial cells cysteinyl leukotriene-induced P-selectin expression is mediated via a mechanism independent of the classically defined CysLT₁ receptor.

	Methods

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Endothelial cell culture. Endothelial cells were isolated from human umbilical veins by digestion with 0.2% collagenase according to the method of Jaffe et al. (1973). The cell suspension was seeded in 25-cm² tissue culture flasks (Nunc Inc., Naperville, IL) coated with 0.5% gelatin and 25 µg/ml fibronectin. Cells were cultured in M199 supplemented with 20% normal human serum, L-glutamine (2 mM), penicillin (196 U/ml), streptomycin (196 µg/ml) (complete M199) and 25 µg/ml endothelial cell growth supplement. Cultures were allowed to grow to confluence at 37°C under 5% carbon dioxide and culture media were changed every 3 days. For adhesion experiments HUVEC at first passage were plated on eight-chamber Lab Tek slides (Nunc Inc.) coated with 0.5% gelatin and 25 µg/ml fibronectin. Cells were seeded at 90 to 95% confluence and used upon reaching visual confluence, typically within 24 h. Endothelial cells were characterized by a cobblestone appearance, and all experiments were performed with cells that had been passaged only once.

Detection of P-selectin expression on the endothelial cell surface. Detection of surface expression of P-selectin on cultured endothelial cells was performed by modification of a method described previously (Birch et al., 1994). Polyclonal goat antimouse IgG-coated magnetic, 4.5 µm, polystyrene beads (Dynal Inc, Great Neck, NY) were secondarily coated with either an anti-P-selectin mAb (GA6, Becton Dickinson, San Jose, CA) or with an irrelevant murine IgG₁ mAb (Coulter Corp., Hialeah, FL) as a control. Beads were coated with the appropriate antibody by incubation in DPBS containing 0.25% HSA overnight at 4°C with gentle rotation at a final concentration of 2.8 µg antibody/mg beads. Beads were then washed a total of four times for 30 min each with DPBS + 0.25% HSA at 4°C and then resuspended in DPBS containing 0.2% HSA (DPBS + 0.2% HSA) such that each well to be assayed received 300 µl of antibody-coated bead suspension (final concentration, 1.5 mg beads/ml). The bead suspension was mixed well and allowed to warm to room temperature (22°C).

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Isolation of human lung tissues. Macroscopically normal human lung tissue was obtained from two patients undergoing surgical resection and four organ donors (supplied by the International Institute for the Advancement of Medicine, Exton, PA or the Anatomical Gift Foundation, Woodbine, GA). The surgical specimens were from patients with lung carcinoma, whereas the organ donor specimens (mean age, 23.5 ± 5.5 years; three males, one female) were mainly from victims of head trauma (gun shot wounds, motor vehicle accidents, closed head injury). Surgical specimens were placed in RPMI 1640 solution at 4°C within 90 min of resection for the 15-min transfer to the laboratory. Organ donor specimens were placed in cooled (4°C) tissue preservation solution (Viaspan; Du Pont Merck Pharmaceutical Co., Wilmington, DE) and transferred to the laboratory overnight. On reaching the laboratory all tissues were immediately placed in 4 liters of modified Krebs' bicarbonate solution of the following composition (mM): NaCl, 118; KCl, 5.4; NaH₂PO₄, 1.0; MgSO₄, 1.2; CaCl₂, 1.9; NaHCO₃, 25; and D-glucose, 11.1 and gassed with 95% oxygen and 5% carbon dioxide at 4°C. Bronchi (internal diameter, 1-3 mm) were dissected free of surrounding parenchymal lung tissue and were either used immediately or placed in oxygenated Krebs' solution at 4°C for use the following day. No differences were evident in responses to treatments between tissues that were used immediately and those used within the following 40 h.

Organ bath studies. Bronchi were prepared as rings 4 to 5 mm in length and placed over stirrups made of tungsten wire. These were then inserted over straight tungsten pins suspended in 10-ml organ baths containing Krebs' solution maintained at 37°C and gassed with 5% carbon dioxide in oxygen. Tissues were connected to a Grass FT03C force-displacement transducer for the measurement of isometric tension which was recorded on a Grass Model 7 polygraph (Grass Instruments, Quincy, MA). Bronchial preparations were suspended under an initial tension of 1 g and maintained at the same tension. Tissues were washed with fresh buffer every 15 min for a 60-min equilibration period after which the preparations were contracted with histamine (3 µM). When the response had reached a plateau, tissues were washed every 15 min with fresh Krebs' solution and the tissues allowed to return passively to their initial resting tone. Adjacent rings from the same airway tissue were used as control or treated rings. Cumulative concentration-effect curves were obtained for each agonist (LTC₄, LTD₄ or LTE₄). Only one cumulative concentration-effect curve was obtained for each ring preparation. At the end of the concentration-effect curve tissues were exposed to carbachol (1 mM) to elicit maximum contraction.

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Data analysis. All numerical data are expressed as arithmetic mean ± S.E.M. The n values represent the number of separate experiments carried out with cells or lung tissue obtained from different donors. Estimates of total bead adherence to HUVEC monolayers were determined as detailed above. Background levels of bead adherence were determined by measuring the density of antibody-labeled beads over fields from control cells. In each experiment the mean background level of bead density was subtracted from each of the respective means for bead densities obtained over cultures of treated cells. The log (M) EC₅₀ values were determined as the log M concentration of the agonist that produced 50% of maximum bead adherence in each concentration-effect curve. These were converted to the negative logarithm before statistical analysis. It should be noted that in the context of the present study EC₅₀ values for bead adherence were calculated with the assumption that the maximal response was that obtained with the largest concentration of agonist tested, that is, 10⁶ M for LTC₄ and LTD₄ and 10⁵ M for histamine. In studies of isolated human bronchi, log (M) EC₅₀ values were determined as the log M concentration of the agonist that produced 50% of the maximum contraction in each concentration-effect curve. Differences in the log (M) EC₅₀ values for agonists alone were evaluated with use of the Student's t test for unpaired observations, whereas differences in the log (M) EC₅₀ values for between agonists in the absence and presence of selective receptor antagonists were evaluated with use of the Student's t test for paired observations. Probability values < .05 were considered significant.

Reagents. RPMI 1640, M199, L-glutamine, penicillin and streptomycin were obtained from Gibco BRL (Gaithersburg, MD); endothelial growth supplement was from Collaborative Biomedical Products (Bedford, MA); collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ); DPBS was from Biofluids (Rockville, MD); normal human serum, HSA, fibronectin, histamine diphosphate salt and pyrilamine maleate were purchased from Sigma Chemical Co. (St. Louis, MO); LTC₄, LTD₄, LTE₄ and LTB₄, SKF 104353, pranlukast, zafirlukast and burimamide were a generous gift from SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Stock solutions (10² M) of leukotrienes were previously prepared by dissolving LTC₄ in methanol and LTD₄ or LTE₄ in ethanol. These were aliquoted and stored at 70°C until used. SKF 104353, pranlukast and zafirlukast were prepared by dissolving these compounds in distilled water, dimethyl sulfoxide and 0.1 N sodium hydroxide, respectively. All other drugs were dissolved in DPBS. Subsequent dilutions of drugs and stock solutions were made in DPBS with all drug dilutions prepared fresh on the day of experimentation.

	Results

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Effect of cysteinyl leukotrienes on P-selectin expression in HUVEC and contraction of airway smooth muscle preparations. Exposure of human endothelial cells to LTC₄ or LTD₄ for 10 min caused a concentration-related increase in the adherence of anti-P-selectin labeled beads to HUVEC monolayers (fig. 1a). This concentration-related increase in binding of beads to stimulated HUVEC was evident both by determination of the density of beads bound per field of cells and was also readily observable by phase-contrast microscopy. After exposure of HUVEC to 10⁶ M LTC₄ mean bead density was 332.4 ± 31.7 beads/field (average of eight fields from each of n = 6 separate experiments). In control cultures which received the vehicle for LTC₄ in place of agonist, mean bead density was only 4.1 ± 1.2 beads/field (average of eight fields from each of n = 6 separate experiments). Extremely low levels of bead adherence to HUVEC were also observed in cultures where cells were stimulated with LTC₄ at 10⁶ M and then exposed to beads which were either unlabeled or labeled with the irrelevant murine IgG₁ mAb. Under these conditions mean bead density was found to be 2.4 ± 0.9 and 2.7 ± 0.6 beads/field, respectively (average of eight fields from each of n = 3 separate experiments).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Effect of LTC4 (), LTD4 () and LTE4 () on P-selectin expression in cultured human endothelial cells (a) and contraction of airway smooth muscle in isolated human bronchi (b). (a) Confluent monolayers of HUVEC were incubated with the indicated concentrations of LTC4, LTD4 or LTE4 for 10 min at 37°C and surface expression of P-selectin was detected by means of polystyrene beads coated with an anti-P-selectin mAb as described under "Methods." Maximal responses to LTC4 and LTD4 at 106 M were 332.4 ± 31.7 beads/field and 171.0 ± 26.9 beads/field, respectively; whereas at 106 M LTE4, mean bead density was only 3.6 ± 1.4 beads/field. Responses are expressed as beads bound per field of endothelial cells. (b) Isolated bronchial preparations were incubated with the indicated concentrations of LTC4 (), LTD4 () or LTE4 () and contractions are expressed as a percent of carbachol (1 mM) responses in these tissues. All values are the mean ± S.E.M. For endothelial cells, n = 6 for LTC4 and LTD4 and n = 5 for LTE4. For lung samples, n = 6. Mean bead density significantly different from that obtained for LTC4 at the same agonist concentration, * P < .05 and ** P < .01.

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Effect of CysLT₁ receptor antagonists on leukotriene-induced responses in HUVEC and isolated airway smooth muscle preparations.. The effects of three structurally unrelated selective CysLT₁ receptor antagonists, SKF 104353, pranlukast and zafirlukast, on LTC₄-induced P-selectin expression in HUVEC are shown in figure 2. The concentration of each antagonist used was 10⁵ M, which is some 1000 times greater than their K_d values for the CysLT₁ receptor (Hay et al., 1987; Buckner et al., 1990; Fujiwara et al., 1993). Thus, this concentration of antagonist would be predicted to cause a greater than 3 log-fold shift to the right of the agonist dose-response curve if CysLT₁ receptors were involved. However pretreatment of HUVEC with either pranlukast, zafirlukast or SKF 104353 failed to significantly modify the adherence of anti-P-selectin labeled beads induced by LTC₄ (fig. 2, a, b and c). Neither the maximal response nor the log (M) EC₅₀ values (taking the response to 1 µM as a maximal response) for LTC₄ were altered in the presence of these antagonists. As illustrated in figure 2, in all cases the LTC₄ concentration-effect curves in the absence and presence of antagonist were superimposable. By contrast, pretreatment of isolated human airway preparations with a 10-fold lower concentration of SKF 104353 significantly antagonized the contractile effect of LTC₄, which caused the expected 3 log-fold rightward shift in the agonist dose-response curve (fig. 2e).

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of selective CysLT1 receptor antagonists on cysteinyl leukotriene-induced P-selectin expression in cultured human endothelial cells and contraction of human isolated bronchi. HUVEC were incubated with the indicated concentration of LTC4 in the absence () or presence of (a) 105 M pranlukast (); (b) 105 M zafirlukast (); (c) 105 M SKF 104353 (); and surface expression of P-selectin was detected by means of polystyrene beads coated with an anti-P-selectin mAb as described in figure 1a. Bead density is expressed as a percent of the maximum LTC4 (106 M) responses in these cultures. Maximal responses for LTC4 were: control, 310.5 ± 27.6 beads/field; pranlukast, 320.0 ± 53.2 beads/field; zafirlukast, 320.6 ± 46.8 beads/field; and SKF 104353, 300.8 ± 37.0 beads/field. The log M concentrations of LTC4 causing 50% of the maximum response (taking the response at 1 µM LTC4 to be the maximum; see "Data Analysis") were: control, 7.00 ± 0.14; pranlukast, 6.95 ± 0.24; zafirlukast, 6.91 ± 0.20; and SKF 104353, 6.86 ± 0.17, in paired samples from five separate experiments. (d) Confluent monolayers of HUVEC were incubated with the indicated concentration of LTD4 in the absence () or presence () of (105 M) SKF 104353, with bead density expressed as a percent of the maximum LTD4 (106 M) responses in these cultures. Maximal responses for LTD4 were: control, 154.7 ± 30.0 and SKF 104353, 154.2 ± 22.4 beads/field. (e) Isolated bronchial preparations were incubated with LTC4 in the absence () or presence () of (106 M) SKF 104353 and contractions are expressed as a percent of carbachol (1 mM) responses in these tissues. All values are the mean ± S.E.M. with n = 5 for endothelial cells and n = 6 for lung samples.

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View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of selective histamine receptor antagonists on histamine-induced P-selectin expression in cultured human endothelial cells. HUVEC were incubated with histamine in the absence () and presence of the histamine H1 receptor antagonist pyrilamine (105 M, ) or the histamine H2/H3 receptor antagonist burimamide (105 M, ]), and surface expression of P-selectin was detected by means of polystyrene beads coated with an anti-P-selectin mAb as described in figure 1a. Maximal responses for histamine were: control, 377.2 ± 42.6; pyrilamine, 6.7 ± 2.7; and burimamide, 350.9 ± 45.2. Bead density is expressed as a percent of the maximum histamine (105 M) responses in these cultures. Data points represent the mean ± S.E.M., n = 3. * Responses significantly different from those induced by histamine alone, P < .05.

	Discussion

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In the present study we have shown that the cysteinyl leukotrienes LTC₄ and LTD₄ induce surface expression of P-selectin in HUVEC. Comparison of the responses obtained with LTC₄ and histamine show that LTC₄ was about 10-fold more potent than and equally efficacious as histamine, a mediator well known to induce surface expression of endothelial P-selectin (Hattori et al., 1989a; McEver et al., 1989; Lorant et al., 1991). The findings that LTC₄ and LTD₄ cause surface expression of P-selectin in HUVEC are consistent with previous reports demonstrating that LTC₄ and LTD₄ induce expression of P-selectin in cultured human endothelial cells (Datta et al., 1995; Papayianni et al., 1996). These data, together with the observations that cysteinyl leukotrienes cause adhesion of leukocytes to endothelial cell monolayers in vitro (McIntyre et al., 1986; Papayianni et al., 1996) and accumulation of inflammatory cells in vivo (Foster and Chan, 1991; Laitinen et al., 1993), suggest that these mediators may play a role in leukocyte recruitment during inflammation.

In the present study surface expression of endothelial P-selectin was detected through the use of small polystyrene beads coated with an anti-P-selectin mAb. This method not only allowed for ligand detection but also lent itself to quantitation of the response by determining the density of beads adherent to cultured endothelial cells (Birch et al., 1994; Datta et al., 1995). Stimulation of human endothelial cells with either LTC₄ or LTD₄ produced surface expression of P-selectin which was rapid and concentration dependent. Under the present conditions LTC₄ was found to be more effective than LTD₄ at inducing P-selectin expression, whereas LTE₄ and LTB₄ were ineffective. This pattern of response for LTC₄ and LTD₄ was similar to that observed by Papayianni and co-workers (1996), who used a radiolabeled secondary antibody to detect endothelial P-selectin; it is also similar to the order of potency reported for the ability of LTC₄ and LTD₄ to cause another endothelial-related event, namely the secretion of von Willebrand factor from HUVEC (Datta et al., 1995). Both LTC₄ and LTD₄ were shown to induce eosinophil recruitment into guinea pig airways in vivo, whereas LTE₄ and LTB₄ were ineffective (Foster and Chan, 1991), although the site of action of the cysteinyl leukotrienes in this animal model is unknown.

The activity profile of the cysteinyl leukotrienes with respect to endothelial P-selectin expression contrasts with the effectiveness of these agonists in causing contraction in isolated human airway smooth muscle preparations. In these latter tissues, LTC₄ and LTD₄ produce similar dose-response curves as shown in this study and by others (Buckner et al., 1986, 1990; Labat et al., 1992). In isolated human bronchi the actions of cysteinyl leukotrienes to cause airway smooth muscle contraction have been attributed to activation of the CysLT₁ receptor. In this tissue preparation the contractile effects of cysteinyl leukotrienes are readily blocked by selective CysLT₁ receptor antagonists (Buckner et al., 1986; Hay et al., 1987; Labat et al., 1992; Fujiwara et al., 1993). The characteristics of the receptor responsible for cysteinyl leukotriene-induced P-selectin expression in human endothelial cells are unclear, however. In our investigation we used three structurally unrelated, potent and selective CysLT₁ receptor antagonists. The observation that, at concentrations 1000-fold greater than their estimated K_d values for the CysLT₁ receptor, SKF 104353, pranlukast and zafirlukast failed to inhibit the LTC₄- or LTD₄-induced P-selectin expression suggests that the agonists were acting by a mechanism independent of the CysLT₁ receptor. Thus as graphically depicted in figure 2, it appears that the leukotriene receptor subtype responsible for LTC₄- and LTD₄-induced P-selectin expression in human endothelial cells is different from that which mediates leukotriene-induced human airway smooth muscle contraction. Although the characteristics of the receptor responsible for mediating cysteinyl leukotriene-induced P-selectin expression are unclear, within the current system results obtained for the histamine receptor antagonists are consistent with the hypothesis that histamine acts via the classical H₁ receptor subtype to evoke endothelial P-selectin expression (Kubes and Kanwar, 1994).

Experiments with isolated human and animal tissues have already provided evidence for the existence of more than one leukotriene receptor (Fleisch et al., 1982; Snyder and Krell, 1984; Labat et al., 1992; Gardiner et al., 1994). Several studies with leukotriene receptor antagonists have demonstrated that there are least two cysteinyl leukotriene receptor types, one sensitive to a range of leukotriene antagonists and another which is insensitive to such compounds (Snyder and Krell, 1984; Gardiner et al., 1990, 1994; Cuthbert et al., 1991). In a comparative study on the contractile effects of cysteinyl leukotrienes in human airway and pulmonary vein smooth muscle it was reported that there were two types of leukotriene receptors in human lung, one found on airways which was inhibited by CysLT₁ antagonists and a second found on pulmonary veins which was resistant to the CysLT₁ antagonists (Labat et al., 1992). The antagonist data presented in the current study are in accord with such findings.

It should be noted that leukotriene receptor antagonists have been shown to be effective against cysteinyl leukotriene-induced endothelial responses. In two studies with cultured human endothelial cells, SKF 104353 was shown to inhibit LTD₄-induced secretion of von Willebrand factor (Datta et al., 1995) and the LTC₄- or LTD₄-induced increase in endothelial cell cytosolic calcium (Heimbürger and Palmblad, 1996). However in both these cases high concentrations of the antagonist (1 µM) were required for effect. In addition, CysLT₁ antagonists are also effective at inhibiting the increase in microvascular permeability induced by cysteinyl leukotrienes in animal models (Nakagawa et al., 1992; Bochnowicz and Underwood, 1995). In certain tissues such as the guinea pig trachea (Snyder and Krell, 1984) and ileum (Gardiner et al., 1990) it appears that more than one type of functional leukotriene receptor is present. Whether such a situation exists in endothelial cells is unknown.

The identification of cysteinyl leukotrienes as mediators that may play a role in the pathogenesis of various disorders has resulted in the development of strategies to attenuate the biological actions of these agents. Studies with isolated human airway smooth muscle which show that cysteinyl leukotriene-induced contractions could be inhibited by selective leukotriene receptor antagonists has lead to the development of a range of compounds whose antagonist activity profiles are based on their effectiveness in this tissue preparation. It is now apparent that such antagonists are not effective against all cysteinyl leukotriene-induced responses, nor are the actions of cysteinyl leukotrienes limited to effects in airway smooth muscle. It was the major finding of this study that the cysteinyl leukotrienes LTC₄ and LTD₄ induce surface expression of P-selectin in human endothelial cells via a mechanism that appears independent of the classical CysLT₁ receptor. More specific information concerning the nature of the CysLT receptor mediating endothelial P-selectin expression must await better pharmacological and molecular biological tools. Nevertheless, it can be hypothesized that the development of selective antagonists for this putative receptor subtype may have a therapeutic advantage over selective CysLT₁ receptor antagonists in inhibiting some of the proinflammatory effects of cysteinyl leukotrienes.