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The Journal of Immunology, 1999, 163: 3449-3458.
Copyright © 1999 by The American Association of Immunologists

Role of 5-Lipoxygenase Products in the Local Accumulation of Neutrophils in Dermal Inflammation in the Rabbit1

Sylvie Marleau2,3,*, Bernard Fruteau de Laclos{dagger}, Ana B. Sanchez*, Patrice E. Poubelle* and Pierre Borgeat*

* Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du Centre Hospitalier Universitaire de Québec (Pavillon Centre Hospitalier de l’Université Laval) et Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada; and {dagger} Hôpital du Saint-Sacrement, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 
Studies were undertaken to define the role of 5-lipoxygenase (5-LO) products and, in particular, of leukotriene (LT) B4 in the polymorphonuclear leukocyte (PMN) emigration process using a rabbit model of dermal inflammation. Our results show that i.v. administration to rabbits of MK-0591, a compound that inhibits LT biosynthesis in blood and tissues when administered in vivo, significantly reduced 51Cr-labeled PMN accumulation in response to intradermally injected chemotactic agonists, including IL-8, FMLP, C5a, and LTB4 itself. In addition, pretreatment of the labeled PMN with MK-0591 ex vivo before their injection in recipient animals was equally effective in reducing 51Cr-labeled PMN emigration to dermal inflammatory sites. These results support a role for de novo synthesis of 5-LO metabolites by PMN for their chemotactic response to inflammatory mediators. Other studies demonstrated that elevated intravascular concentration of LTB4 interferes with PMN extravasation inasmuch as a continuous i.v. infusion of LTB4, in the range of 5–300 ng/min/kg, dose-dependently inhibited extravascular PMN accumulation to acute inflammatory skin sites elicited by the chemoattractants LTB4, FMLP, C5a, and IL-8 and by TNF-{alpha}, IL-1ß, and LPS; such phenomena may constitute a natural protective mechanism from massive tissue invasion by activated PMN in specific pathologic conditions such as ischemia (and reperfusion). These studies demonstrate additional functions of 5-LO products in the regulation of PMN trafficking, distinct from the well-characterized chemotactic activity of LTB4 present in the extravascular compartment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 
The molecular events that drive migration of polymorphonuclear leukocytes (PMN)4 to sites of inflammation are triggered by the local release of a variety of chemotactic and stimulatory factors. These factors include chemokines, cytokines, complement-derived protein fragments, lipid mediators, and bacterial products, such as formylated peptides that coordinate cytoskeletal rearrangements and adhesive changes essential for effective cell motility. Invading PMN are themselves a rich source of bioactive lipids, including oxygenated derivatives of arachidonic acid that are generated through the 5-lipoxygenase (5-LO) pathway and that may serve as both intracellular and extracellular mediators (1). A well-investigated group of these arachidonic acid derivatives are the leukotrienes (LT). For example, LTB4, after its ligation to high-affinity receptors, is a potent stimulator of PMN chemokinesis and chemotaxis in vitro (2, 3, 4). Early in vivo studies show LTB4 to promote PMN adhesion, diapedesis, and accumulation into tissues when applied extravascularly (5, 6, 7), as well as PMN-dependent plasma leakage (8). Such biologic effects of LTB4, coupled with its release at sites of inflammation, point to a central role of LTB4 as a regulator of cell trafficking.

In support of the important role of LTB4 in inflammation, inhibitors of 5-LO product biosynthesis and LTB4 receptor antagonists have proven to be beneficial in different experimental models of inflammation (9, 10, 11, 12) and in some pathologic conditions (13, 14); for instance, a selective LTB4 receptor antagonist showed a striking efficacy in a murine model of rheumatoid arthritis (12). It is unclear, however, whether these beneficial effects of 5-LO inhibitors and LTB4 antagonists are due to the reduction of the biosynthesis (by PMN) and/or the action of LTB4 at the level of the blood-endothelial interface or to the reduction of LTB4 levels in the extravascular compartment where secreted LTB4 acts as a chemoattractant. Indeed, the reduction of neutrophil infiltration by agents that inhibit the production (or action) of 5-LO metabolites may be related to critical autocrine or paracrine roles of these metabolites acting in the intravascular compartment either on the PMN themselves or on the endothelial cells (15, 16) and regulating adhesion and movement of PMN. Experimental evidence suggesting the importance of LTB4 and/or LTA4 biosynthesis by the PMN themselves for PMN responsivity originates from observations that zymosan-activated serum- and IL-8-induced chemotaxis in vitro was prevented by pretreatment of PMN with 5-LO product biosynthesis inhibitors, thereby suggesting that chemoattractant-induced recruitment of PMN implies PMN 5-LO activation (1). In addition to providing LTB4 for autocrine activation of PMN or paracrine stimulation of endothelial cells, agonist-induced 5-LO activation in circulating PMN may also provide LTA4 for transcellular metabolism (17, 18), allowing LTB4, LTC4, and lipoxin biosynthesis by other cells, including platelets and endothelial cells (19, 20). The potent vasoactive compound LTC4 increases vascular permeability and endothelial cell hyperadhesiveness for PMN (21), although by a mechanism different from that of LTB4 (15, 16); in contrast, lipoxin A4 has been shown to block PMN migration triggered by LTB4 (22). Thus, amplification of the production of LTs and lipoxins at the blood-endothelium interface through transcellular metabolism of LTA4 may further modulate PMN recruitment.

Interestingly, LTB4 may exert either a stimulatory or an inhibitory effect on PMN extravasation, depending on its distribution between the extra- and the intravascular compartments. In pathophysiologic conditions such as hindlimb ischemia and reperfusion, LTB4 has been found to accumulate in plasma (23). In these circumstances, the chemotactic response of circulating PMN toward ischemic plasma (containing elevated levels of LTB4) applied extravascularly is blunted, an effect that is attributed to LTB4 receptor desensitization on exposure of the cells to LTB4 (23). Similarly, the chemotactic hyporesponsiveness to LTB4 of peripheral blood PMN (as assessed by ex vivo chemotactic assays) observed in patients with diseases such as cystic fibrosis has also been attributed to in vivo deactivation of LTB4 receptors as a result of chronic vascular exposure to high local concentration of LTB4 (24). Interestingly, decreased sensitivity of the PMN to other chemotaxins, in addition to LTB4, has also been observed in various diseases or inflammatory situations (25, 26, 27).

The present studies were undertaken to assess whether the cellular events involved in neutrophil extravasation in response to chemoattractants applied extravascularly are dependent on 5-LO activity of circulating PMN. Our results show that either i.v. pretreatment of rabbits or ex vivo pretreatment of 51Cr-labeled PMN with MK-0591, an inhibitor of 5-LO product biosynthesis (28), significantly reduced 51Cr-labeled PMN accumulation in response to locally injected chemotactic agents. Furthermore, to clarify the effect of sustained exposure of PMN to intravascular LTB4 on their migratory responses, we examined the effect of i.v. infusions of LTB4 on neutrophil emigration induced by local injections of various chemoattractants in vivo. Our data indicate that continuous exposure of circulating PMN to steady-state concentrations of LTB4 results in a nonselective, dose-dependent inhibition of their migratory response to a variety of chemotactic stimuli.


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

5(S),12(R)-Dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) and the 5-LO product synthesis inhibitor MK-0591 were generously provided by Drs. R. Young and J. Mancini (Merck Frosst Centre for Therapeutic Research, Pointe-Claire, Québec, Canada). A stock solution of LTB4 (200 µg/ml, in ethanol) was kept at -20°C and diluted in vehicle immediately before use. PGE2, FMLP, A23187 (free acid), BSA (low endotoxin), LPS from Escherichia coli (serotype 026:B6), human recombinant (hr) C5a, human myeloperoxidase (MPO), hexadecyltrimethylammonium bromide, and 3,3',5,5'-tetramethylbenzidine were purchased from Sigma (St. Louis, MO). Dextran T-500 and Percoll were obtained from Pharmacia Canada (Baie d’Urfé, Québec, Canada), and HBSS and HEPES from Life Technologies (Grand Island, NY). hrTNF-{alpha} was kindly provided by Genentech (South San Francisco, CA), and hrIL-8 was a generous gift from Dr. Henry Showell (Pfizer Pharmaceuticals, Groton, CT). 51Cr (250–500 mCi/mg) was obtained from Frosst (Division of Merck Frosst Canada, Kirkland, Québec, Canada). All solutions for parenteral administration were made from pyrogen-free, sterile 0.9% NaCl containing 5% dextrose (Baxter Travenol Laboratories, Malton, Ontario, Canada).

Animals

Male New Zealand White rabbits (2.3–3.5 kg) were purchased from Charles River (St-Constant, Québec, Canada). They were maintained in individual cages with free access to food (Purina pellets) and water for at least 5 days before any experimental work was undertaken. At the completion of each experiment, the animals were killed with an overdose of pentobarbital (Euthanyl, MTC Pharmaceuticals, Canada Packers, Cambridge, Ontario, Canada).

Cell separation and radiolabeling procedures

Peripheral rabbit PMN were isolated according to Haslett et al. (29). Rabbit blood (32 ml) was collected from the carotid artery into 50-ml polypropylene tubes containing 8 ml of a 3.8% trisodium citrate solution and centrifuged at 300 x g for 20 min. Platelet-rich plasma was removed and centrifuged at 2000 x g for 20 min over a 3-ml Percoll cushion (90% Percoll in 0.9% saline) to obtain platelet-poor plasma (PPP). After mixing the remaining buffy coat and erythrocyte layer with 8 ml of 6% dextran T-500 in 0.9% NaCl and adjusting the volume to 40 ml, the erythrocytes were allowed to sediment at room temperature for 30 min. The leukocyte-rich layer was then carefully removed and centrifuged at 275 x g for 8 min. The cell pellet was resuspended in 4 ml of PPP, overlayered onto a discontinuous plasma Percoll gradient (43% and 53% Percoll in PPP), and centrifuged at 260 x g for 11 min. The neutrophil-rich band was removed, mixed with 2 ml of PPP, and separated in three aliquots. To remove contaminating erythrocytes, 20 ml of cold (4°C) 0.2% NaCl was added to each aliquot and left 10 s before addition of 20 ml of 1.6% NaCl/10 mM dextrose. The cells were then centrifuged at 170 x g for 8 min and resuspended into HBSS-HEPES (10 mM), pH 7.4, containing 10% PPP. The aliquots were pooled, and the cells were labeled with 51Cr by incubating with 200–250 µCi of Na251CrO4 at 37°C for 60 min. The cells were then washed twice in HBSS-10% plasma and resuspended into 2 ml of plasma before injection into animals.

Measurement of 51Cr-labeled neutrophil accumulation in rabbit skin

The day before the experiment, the dorsal region of rabbits was shaved. Anesthesia was induced by an i.m. injection of ketamine (Rogarsetic, Rogar/STB, London, Ontario, Canada) (35 mg/kg), and xylazine (Rompun, Haver, Etobicoke, Ontario, Canada) (5 mg/kg), and the anesthetized animals were placed on heating pads. Two catheters were installed, one in the marginal vein of an ear (PE-50; Clay Adams, Parsippany, NJ) to allow the infusion of ketamine (45 mg/kg/h) in 0.9% NaCl-5% glucose (50:50 v/v) at a rate of 12 ml/h, and one in the central artery of an ear (Butterfly-21; Abbott Ireland, North Chicago, IL) to allow blood sampling. Supplemental injections of xylazine (5 mg/kg i.m.) were given every 1–2 h as needed to maintain anesthesia. Fifteen to 20 min after i.v. injection of 51Cr-labeled PMN (~30 x 106 cells/animal, 1–3 µCi/kg), chemoattractants, including LTB4 (10–500 pmol/site), hrC5a (1–100 pmol/site), FMLP (10–500 pmol/site), hrIL-8 (1–10 pmol/site), hrTNF-{alpha} (0.1–10 pmol/site), and hrIL-1ß (1 pmol/site), were injected intradermally (i.d.) in 0.05 ml of HBSS-HEPES (10 mM)/0.1% BSA, each in four replicates. Where indicated, agonists were coinjected with PGE2 (300 pmol/site) as a vasodilator to potentiate neutrophil extravasation (8). Three blood samples (2 ml) were withdrawn in the course of the experiments to determine the mean concentration of circulating labeled PMN and the percentage of cell-associated and cell-free radioactivity in plasma. The animals were killed by an overdose of pentobarbital, the dorsal skin was removed and cleaned of excess blood, and the injection sites were excised using an 11 mm diameter punch (in some experiments the protocol used was different and two series of skin biopsies were collected directly from the back of the animals; see i.v. infusion of LTB4 below). The specific activity of the labeled cell suspension (51Cr counts per neutrophil) was used to determine the number of labeled PMN per site, which was normalized to 104 circulating labeled PMN per ml of blood.

Intravenous infusion of MK-0591

After a 30-min stabilization period during which the animals were infused with vehicle (5% glucose) and ketamine, animals received a bolus of MK-0591 (2 mg/kg), which was followed by a continuous infusion of MK-0591 (5 µg/min/kg in 5% glucose) to maintain a plasma concentration of the drug of ~1 µM; we had previously shown that this regimen induced a significant inhibition (~80%) of LTB4 biosynthesis in A23187-stimulated whole rabbit blood ex vivo (30). Forty minutes later, agonists, including LTB4, FMLP, C5a, and IL-8, were injected i.d., and 51Cr-labeled PMN were allowed to accumulate over a 1-h test period during MK-0591 infusion.

MK-0591 pretreatment of 51Cr-labeled PMN

51Cr-labeled PMN were incubated with MK-0591 (10-5 M in 10% PPP) or vehicle (DMSO, 1 µl/ml in 10% PPP) for 20 min at room temperature before the final wash, resuspension in PPP, and injection into recipient animals.

Intravenous infusion of LTB4

After a 30-min stabilization period during which the animals were infused with vehicle (0.9% NaCl-5% glucose (50:50 v/v) containing 0.01% BSA) and ketamine, an infusion of LTB4 (5–300 ng/min/kg) or vehicle (<1% ethanol in 0.9% NaCl-5% glucose (50:50 v/v) containing 0.01% BSA) was initiated at a rate of 12 ml/h 30 min before the i.d. injection of agonists as described above. 51Cr-labeled PMN were allowed to accumulate over a 1-h test period during LTB4 infusion. Animals were killed, and the dermal inflammatory sites were excised as described above. In another series of experiments, 51Cr-labeled PMN were allowed to accumulate for 1 h at inflammatory sites in anesthetized animals infused with vehicle only before skin biopsies were excised using a 3 mm diameter punch; LTB4 was then administered either as a bolus (5 µg/kg) or as an infusion (100 ng/min/kg), and a second series of i.d. injections was administered 30 min after the beginning of the LTB4 infusion. The biopsies were excised 1 h later, and the animals were killed with an overdose of pentobarbital. In these experiments, each animal serves as its own control.

Measurement of MPO activity in rabbit skin

In other series of experiments, neutrophil accumulation in response to i.d. injected agonists was estimated by measuring MPO activity in skin biopsies. Agonists (in 0.05 ml of 0.9% NaCl; four replicates) were injected twice, first at t = 0 (at the beginning of an infusion of vehicle) and secondly at t = 3.5 h (30 min after initiation of an infusion of vehicle or LTB4, in 50/50 (v/v) 0.9% NaCl/5% glucose solution containing ketamine and <1% ethanol); two series of biopsies were excised 3 h after the i.d. injections of chemoattractants. The rabbits were anesthetized throughout the experiments and killed with an overdose of pentobarbital at the end of the experiment. The agents under investigation included LTB4 (300 pmol/site) and one of the following agents: IL-8 (100 pmol/site), TNF-{alpha} (300 pmol/site), LPS (500 ng/site), C5a (100 pmol/site), and FMLP (240 pmol/site). In this particular series of experiments, as well as in the studies of the generation of LTB4 in the skin (see below), each agent was injected i.d. together with PGE2 (300 pmol/site), a vasodilator, to enhance PMN migration (8, 31) and facilitate measurements of MPO activity in the 3 mm diameter skin biopsies. After excision, the skin biopsies were immediately frozen in liquid nitrogen and kept at -70°C until assayed for MPO. Briefly, the biopsies were thawed, weighed, and homogenized in 1 ml of potassium phosphate buffer, pH 5.4, containing 0.5% hexadecyltrimethylammonium bromide. The homogenates were sonicated on ice for 15 s, frozen (20 min at -70°C), thawed, sonicated again, and centrifuged (15°C, 2700 x g for 25 min). A 16 mM stock solution (50 µl) of 3,3',5,5'-tetramethylbenzidine (in DMSO) was added to 100 µl of supernatant and 100 µl of buffer, and the solutions were preincubated at 37°C for 5 min. The enzymatic reaction was started by the addition of 250 µl of a solution of 0.38 mM H2O2 in 0.08 M phosphate buffer, pH 5.4, and incubated for 10 min. The reaction was terminated by the addition of catalase (50 µl of a 200 µg/ml solution in PBS), followed 3 min later by the addition of 1 ml of 0.2 M sodium acetate (pH 3.0). The absorbance of the samples and of MPO standards (0.0625–1.0 U/ml) were determined at 655 nm. The results are expressed as U/g of tissue and normalized to 106 PMN per ml of blood.

Generation of LTB4 in the skin

FMLP (240 pmol/site) and C5a (100 pmol/site), both coinjected with PGE2 (300 pmol), were tested for their ability to stimulate LTB4 biosynthesis in skin. Skin biopsies were punched out at 1, 5, 30, 90, and 180 min after the i.d. injections using a 3 mm diameter punch. Skin biopsies were immediately frozen in liquid nitrogen and kept at -70°C until assayed for LTB4. In additional experiments, MK-0591 (2 mg/kg i.v. bolus, followed by a continuous infusion of 5 µg/min/kg) was administered 40 min before the i.d. injections of FMLP and C5a.

Effect of MK-0591 on LTB4 biosynthesis in vitro

Rabbit PMN (5 x 106/ml in HBSS containing 10 mM HEPES and 10% PPP) were incubated for 15 min at room temperature in the presence of MK-0591 (10-5 M, final) or vehicle (1 µl DMSO/ml). The cells were washed, resuspended in PPP, and incubated at 37°C for various times up to 150 min. The cells were then washed and resuspended in HBSS/10 mM HEPES, pH 7.4, containing 1 mM calcium and either primed with a mixture of TNF-{alpha} (100 U/ml) and LPS (1 µg/ml) for 20 min at 37°C or directly stimulated with 1 µM A23187 for 5 min at 37°C. The primed cells were stimulated with 300 nM FMLP and platelet-activating factor for 10 min. The reaction was stopped by adding cold (4°C) methanol solution containing PGB2 and 19-hydroxy-PGB2 as internal standards.

Analysis of LTB4

LTB4 was extracted from skin biopsies in methanol (containing 19-hydroxy-PGB2 and PGB2); each biopsy was left in 2 ml of methanol overnight at -20°C. The recovery of LTB4 from skin biopsies (84 ± 3%) has been assessed by estimating the recovery of [3H]LTB4 injected into excised skin biopsies (n = 19). LTB4 concentration in methanol extracts was determined by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) after HPLC purification of the samples, as described previously (30). Briefly, the HPLC fractions containing LTB4 were collected and evaporated under reduced pressure with a Speed Vac concentrator (Savant Instruments, Hicksville, NY), and the dried residues were dissolved with 50 µl of ethanol and diluted by addition of 1 ml of enzyme immunoassay buffer (32).

Statistical analysis

All results are expressed as mean ± SEM, and statistical comparisons were performed by one- or two-way ANOVA where appropriate, unless otherwise indicated. The minimal level of significance was considered as p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Aknowledgments
 References
 
Effects of i.v. infusion of MK-0591 on inflammatory mediator-induced 51Cr-labeled neutrophil accumulation and LTB4 biosynthesis in rabbit skin

Intradermal injections of LTB4, FMLP, C5a, and IL-8 induced a dose-dependent accumulation of 51Cr-labeled PMN over the 1-h test period in rabbit skin (Fig. 1Go and Fig. 3Go). The number of labeled PMN accumulating in the skin was significantly reduced in animals treated with MK-0591 40 min before the i.d. injections (41, 54, 52, and 30% inhibition for i.d. injections of 10 pmol/site of LTB4, FMLP, C5a, and IL-8, respectively) (Fig. 1Go). The biosynthesis of LTB4 in skin biopsies was rapidly and transiently increased upon i.d. injection of either FMLP (240 pmol/site) and C5a (100 pmol/site) (Fig. 2Go), with this effect being greatly diminished after systemic pretreatment with MK-0591 (Table IGo).



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  		FIGURE 1. Effect of infusion of MK-0591 on 51Cr-labeled PMN accumulation in skin in response to i.d. injections of inflammatory mediators (0.05 ml/site). LTB4, FMLP, hrC5a, and hrIL-8 were injected in HBSS containing 10 mM HEPES and 0.1% BSA, in four replicates. Results are the mean ± SEM of responses in six pairs of animals and are expressed as the number of 51Cr-labeled PMN per skin site, normalized to 104 circulating labeled PMN per ml of blood. A significant difference between responses to i.d. injected agonists in the vehicle-treated and MK-0591-treated animals is shown: *, p < 0.05; and **, p < 0.01. Solid and dashed horizontal lines show levels of 51Cr-labeled PMN per site determined after i.d. injections of vehicle (HBSS containing 10 mM HEPES and 0.1% BSA) in the vehicle-treated and MK-0591-treated animals, respectively.

 


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  		FIGURE 3. Effect of MK-0591 pretreatment of 51Cr-labeled PMN ex vivo on their accumulation in skin in response to i.d. injections of inflammatory mediators in vivo. LTB4, FMLP, hrC5a, and hrIL-8 were injected in HBSS containing 10 mM HEPES and 0.1% BSA, in four replicates. Results are the mean ± SEM of responses in six pairs of animals and are expressed as the number of 51Cr-labeled PMN per skin site, normalized to 104 circulating labeled PMN per ml of blood. A significant difference between responses to i.d. injected agonists in the vehicle-treated and MK-0591-treated 51Cr-labeled PMN is shown: *, p < 0.05; and **, p < 0.01. Agonists, injected together with 300 pmol PGE2, are represented by circles. Solid and dashed lines represent levels determined after i.d. injection of vehicle (HBSS containing 10 mM HEPES and 0.1% BSA) in animals receiving the vehicle-treated and MK-0591-treated 51Cr-labeled PMN, respectively.

 


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  		FIGURE 2. Time course of LTB4 generation in skin after i.d. injections of chemoattractants in anesthetized rabbits. FMLP (240 pmol/site) and hrC5a (100 pmol/site) were injected in 50 µl of NaCl 0.9% containing 300 pmol PGE2, in four replicates. Skin biopsies (3 mm) were punched out at 1, 5, 30, 90, and 180 min after the i.d. injections of agonists and were immediately frozen in liquid nitrogen and kept at -70°C until assayed for LTB4. Results are the mean ± SEM in 10 (t = 0 and t = 1 min), 8 (t = 5 and t = 30 min), and 4 (t = 90 and t = 180 min) animals.

 

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  		Table I. Effect of an infusion of MK-0591 on LTB4 biosynthesis in rabbit skin sites upon injection of FMLP and C5aa

 
Effect of ex vivo pretreatment of 51Cr-labeled rabbit PMN with MK-0591 on inflammatory mediator-induced 51Cr-labeled neutrophil accumulation in rabbit skin

Preliminary experiments were performed to assess whether rabbit PMN treated ex vivo with MK-0591 under conditions known to cause complete inhibition of 5-LO product synthesis retain a significant level of inhibition after removal of the drug and prolonged incubation in the presence of PPP. When rabbit PMN were treated with MK-0591 (10-5 M) for 15 min and then washed in HBSS/10 mM HEPES (to remove the drug) and incubated in PPP before activation of LT synthesis with A23187 or natural stimuli (as described in Materials and Methods), LTB4 synthesis was significantly reduced (by 54 ± 4% (n = 3) and 62 ± 2% (n = 3), respectively) in comparison with PMN treated similarly but in the absence of MK-0591. Moreover, the inhibitory effect of MK-0591 on LTB4 biosynthesis measured in cells immediately after removal of the drug or after 2 h of incubation in PPP in absence of the drug was identical, demonstrating that in the experimental conditions used, a part of the inhibitory effect of MK-0591 (50–60%) was irreversible. In the migration studies, ex vivo pretreatment of 51Cr-labeled PMN with MK-0591 significantly reduced their accumulation in the skin (40, 41, 45, and 33% inhibition for i.d. injections of 500 pmol/site of LTB4 and FMLP, 100 pmol/site of C5a and 1 pmol/site of IL-8, respectively) (Fig. 3Go). Pretreatment of 51Cr-labeled PMN with MK-0591 ex vivo did not modify the mean number of labeled PMN circulating in blood (36 ± 12 vs 33 ± 13 (in thousands) 51Cr-labeled PMN/ml of blood in vehicle- and MK-0591-treated cells, respectively).

Effects of i.v. infusion of LTB4 on inflammatory mediator-induced neutrophil accumulation in rabbit skin

Our previous studies have shown that exposure of circulating PMN to steady-state concentrations of LTB4 selectively inhibited the neutropenia induced by i.v. boluses of LTB4, inasmuch as the neutropenia in response to i.v. boluses of FMLP and C5a was retained, a phenomenon that we attributed to a selective desensitization of circulating PMN to LTB4 (32). In initial experiments, we determined whether exposure of circulating PMN to i.v. LTB4 inhibits PMN accumulation into dermal sites injected with LTB4, FMLP, and C5a. As shown in Fig. 4Go, a single i.v. injection of 5 µg LTB4/kg did not prevent 51Cr-labeled PMN emigration in response to LTB4 (300 pmol/site), FMLP (240 pmol/site), and C5a (60 pmol/site), with each agonist being coinjected with 300 pmol PGE2. In contrast, a continuous infusion of 100 ng LTB4/min/kg (an infusion rate that induced a complete desensitization to LTB4 (bolus)-induced neutropenia (32)) significantly reduced the number of labeled PMN that had emigrated in the skin in response to the three agonists (72, 64, and 77% of inhibition relatively to preinfusion values for i.d. injections of LTB4, C5a, and FMLP, respectively).



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  		FIGURE 4. Effect of i.v. administrations of LTB4 on 51Cr-labeled PMN accumulation in skin in response to i.d. injections of LTB4 (300 pmol/site), hrC5a (60 pmol/site), and FMLP (240 pmol/site). Agonists were injected in 50 µl NaCl 0.9% containing 300 pmol PGE2 in four replicates. The dermal accumulation of 51Cr-labeled PMN is expressed as a percentage of values measured for 51Cr-labeled PMN accumulation in agonist-injected skin sites before vehicle infusion or LTB4 administration (bolus or infusion). Results are the mean ± SEM in three (vehicle), four (LTB4 bolus), and eight (LTB4 infusion) animals. *, p < 0.05; **, p < 0.01 compared with preinfusion values.

 
In the next series of experiments, we investigated the dose dependency of the inhibitory effects of i.v. LTB4 infusions on 51Cr-labeled PMN accumulation over infusion rates ranging from 5 to 300 ng LTB4/min/kg (Fig. 5Go). Injections of skin sites with LTB4 (500 pmol) in vehicle-infused animals induced an accumulation of 3.1 ± 1.0 (in thousands) 51Cr-labeled PMN per skin site over a 1-h test period. The amount of labeled PMN accumulating in response to i.d. injected LTB4 was gradually reduced to 1.5 ± 0.4, 1.4 ± 0.2, 0.80 ± 0.11, 0.77 ± 0.19, and 0.61 ± 0.22 (in thousands) 51Cr-labeled PMN per site in rabbits infused with increasing doses of LTB4 (5, 10, 30, 100, and 300 ng/min/kg, respectively) 30 min before and during the 1-h test period (Fig. 5GoA). The dose-dependent inhibitory effect of i.v. LTB4 on 51Cr-labeled PMN emigration extended to a wide variety of chemoattractants including the soluble agonist FMLP (Fig. 5GoB), the chemokine IL-8 (Fig. 5GoC), and the cytokines IL-1ß and TNF-{alpha} (Fig. 5GoD). The relative potency of i.v. LTB4 in reducing 51Cr-labeled PMN accumulation in response to different locally injected chemoattractants was assessed by comparing dose-response curves for approximately equipotent doses of the agonists LTB4 (10 pmol/site), FMLP (10 pmol/site), IL-8 (1 pmol/site), and TNF-{alpha} (10 pmol/site), which induced the accumulation of 1.7 ± 0.5, 2.4 ± 0.7, 1.8 ± 0.6, and 1.2 ± 0.3 (in thousands) 51Cr-labeled PMN per site in vehicle-infused animals, respectively (Fig. 5Go, A–D). The results show that infusion rates of 18, 19, and 20 ng/min/kg are required to reduce by 50% the 51Cr-labeled PMN accumulation in response to i.d. LTB4, FMLP, and IL-8, respectively (Fig. 6Go). In contrast, i.v. LTB4 was less potent at inhibiting 51Cr-labeled PMN emigration in response to i.d. TNF-{alpha} (ID50 of 128 ng LTB4/min/kg) (Fig. 6Go). The inhibitory effect of LTB4 infusion on labeled neutrophil accumulation in vivo was not due to a reduction in the number of circulating 51Cr-labeled PMN in rabbits. At the end of the 1-h in vivo test period, the percentage of circulating 51Cr-labeled PMN was 6.6 ± 0.6% and 8.2 ± 1.1% (mean ± SEM (n = 13) pairs of rabbits) in LTB4 and vehicle-infused animals, respectively.



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  		FIGURE 5. Dose-dependent inhibitory effect of i.v. LTB4 (5–300 ng/min/kg) on 51Cr-labeled PMN accumulation in skin in response to i.d. injections of inflammatory mediators. LTB4 (10 and 500 pmol/site; A), FMLP (10 and 500 pmol/site; B), hrIL-8 (1 and 10 pmol/site; C), and hrIL-1ß and hrTNF-{alpha} (1 and 10 pmol/site, respectively; D) were injected in 50 µl HBSS-HEPES containing 10 mM HEPES and 0.1% BSA in four replicates. LTB4 was infused for a period of 30 min before the i.v. injection of 51Cr-labeled PMN and i.d. injections of inflammatory mediators. At the end of the 60-min migration period, the animals were killed by an overdose of pentobarbital, the back skin was removed, and the injection sites were punched out. Results are the mean ± SEM of replicates and are expressed in terms of the number of 51Cr-labeled PMN per skin site, normalized to 104 circulating labeled PMN per ml of blood. The number of replicates varied between 4 and 13 per infusion rate.

 


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  		FIGURE 6. Relationship between LTB4 infusion rate and the percentage inhibition of dermal 51Cr-labeled PMN accumulation in response to i.d. injections of LTB4, FMLP, and hrTNF-{alpha} (each at 10 pmol/site) and hrIL-8 (1 pmol/site). Data were calculated as percentage inhibition of 51Cr-labeled PMN accumulation on LTB4 infusion vs vehicle infusion (0.9% NaCl-5% glucose (50:50 v/v) containing ketamine). The relative potency of i.v. LTB4 in reducing 51Cr-labeled PMN accumulation was assessed by estimating the ID50 by nonlinear regression analysis using AllFit for Windows. 51Cr-labeled PMN accumulation was reduced by 50% at an infusion rate of 18, 19, 20, and 128 ng LTB4/min/kg in response to i.d. injections of LTB4, FMLP, hrIL-8, and hrTNF-{alpha}, respectively.

 
In parallel experiments, the effect of i.v. infusions of LTB4 on PMN accumulation in skin biopsies was quantitated by determining MPO enzyme activity. Agonists (LTB4, C5a, FMLP, IL-8, and TNF-{alpha}) were injected i.d. at the beginning of an infusion of vehicle. Three hours later, the inflammatory skin sites (3 mm) were excised, immediately frozen in liquid nitrogen, and kept at -70°C until assayed for MPO. After excision of the first series of skin sites, the infusion either was changed to a solution containing LTB4 (100 ng/min/kg) or remained as vehicle only. Thirty minutes later (to allow recovery from the LTB4-induced neutropenia), another series of sites were injected with the same chemoattractants. Three hours later, the second series of skin sites were excised. As shown in Table IIGo, vehicle infusion did not induce a significant change in the MPO activity measured in skin sites after i.d. injections of the agonists when compared with skin sites treated and biopsied before the second infusion. In contrast, infusion of LTB4 significantly reduced the number of PMN accumulating in the skin in response to all agents tested (84, 61, 77, 58, 68, and 66% inhibition for i.d. injections of LTB4, C5a, FMLP, IL-8, LPS, and TNF-{alpha}, respectively) (Table IIGo).


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  		Table II. Effect of an i.v. infusion of LTB4 on mediator-induced neutrophil accumulation in rabbit skin assessed by measurement of MPO activity1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Aknowledgments
 References
 
Our results show that chemoattractants such as C5a and FMLP induce a rapid increase in immunoreactive LTB4 when injected i.d. (Fig. 2Go). Elevation in immunoreactive LTB4 at inflammatory sites was transient, returning to basal levels in ~30 min. In agreement with these data, Aked and Foster (33) reported that LTB4 is cleared rapidly (t1/2 ~ 5 min) from rabbit skin after injection of arachidonic acid. Considering that kinetic studies of 51Cr-labeled PMN accumulation in rabbit skin have shown a maximum rate between 1 and 2 h postinjection, irrespective of the chemoattractant (31, 34), it appears that LTB4 generated at dermal injection sites is derived from resident phagocytes and/or mast cells rather than newly emigrated PMN. Pretreatment (i.v.) of rabbits with MK-0591, in conditions that significantly inhibited the local generation of LTB4 induced by chemoattractants (Table IGo), significantly reduced the number of circulating 51Cr-labeled PMN accumulating in the skin in response to i.d. injections of not only FMLP, C5a, and IL-8, but also of LTB4, which supports the concept that 5-LO activity in the circulating PMN (as opposed to 5-LO activity at the dermal inflammatory sites) contributes to the PMN emigration process (Fig. 1Go). At the dose used, i.v. administered MK-0591 efficiently blocks A23187-stimulated LTB4 synthesis in rabbit whole blood ex vivo (30) as well as in peripheral tissue (Table IGo). To further clarify the relationship between neutrophil 5-LO activity and neutrophil extravascular accumulation in vivo, 51Cr-labeled PMN were preincubated with MK-0591 (and washed to remove the drug) before their injection into recipient animals. This treatment, demonstrated in preliminary experiments to be effective at inhibiting (by ~60%) agonist-induced LTB4 biosynthesis in rabbit PMN in vitro, also significantly reduced 51Cr-labeled PMN accumulation in response to i.d. injected chemoattractants, including LTB4, without affecting the ability of the labeled cells to circulate (Fig. 3Go). Together, these data strongly support the concept that PMN intrinsic 5-LO activity is indeed required for PMN extravasation in the experimental conditions utilized; these data further suggest that the 5-LO activity involved in PMN emigration occurs in the intravascular compartment, likely at the blood-endothelium interface. Indeed, if locally released (extravascular) LTB4 were responsible for 51Cr-labeled PMN accumulation in response to i.d. agonists, then i.d. injection of LTB4 would have restored the emigration of MK-0591-pretreated labeled neutrophils. The partial inhibition of 51Cr-labeled labeled PMN migration observed in our studies is likely related to the only partial inhibition of 5-LO activity in MK-0591-treated cells (see Results). It is also possible that the unlabeled and untreated blood PMN may rescue the 51Cr-labeled and MK-0591-treated PMN through transcellular exchange of LTA4 at the endothelial surface, thus decreasing the impact of MK-0591 pretreatment on the ability of the 51Cr-labeled PMN to generate 5-LO products.

In agreement with our data, Goldman et al. (35) also concluded that an intact lipoxygenase pathway in circulating PMN is essential for their migration; they demonstrated that i.v. pretreatment of rabbits with the lipoxygenase inhibitor diethylcarbamazine prevented PMN accumulation into skin blisters filled either with synthetic LTB4 or with plasma collected from ischemic hindlimbs containing elevated levels of LTB4. However, whether this phenomenon could be generalized to the action of chemically unrelated chemoattractant remained to be determined. Our studies using a highly selective 5-LO product synthesis inhibitor (MK-0591) have clarified this point, by showing that 5-LO inhibition prevents neutrophil accumulation in response to a variety of chemoattractants. Our results are also consistent with those of Guidot et al. (1), who showed that neutrophil 5-LO activity was required for PMN adherence and chemotaxis in vitro, and with those of Bienvenu et al. (36), who showed that PMN-derived LTB4 is likely involved in the increased leukocyte adherence to endothelial cells elicited by low shear rates. Our observations extend these findings by implicating the de novo synthesis of 5-LO metabolites by circulating PMN in mediating neutrophil chemotaxis in response to the local application of a variety of inflammatory mediators, including IL-8, FMLP, C5a, and LTB4 itself. However, it remains to be determined whether the 5-LO metabolites act as intracellular and/or autocrine and paracrine signaling molecules to regulate PMN and endothelial cell functions involved in leukocyte transmigration.

Our results also show that LTB4 may also interfere with PMN extravasation in response to various chemotactic stimuli when circulating cells are exposed to sustained elevated plasma levels of LTB4. Indeed, in contrast to the potent stimulatory effect of extravascular LTB4 on PMN recruitment, continuous intravascular administration of LTB4 resulted in a dose-dependent inhibition of PMN recruitment to inflammatory sites induced in rabbit skin by chemoattractants, IL-1ß and TNF-{alpha}, irrespective of whether the accumulation of 51Cr-labeled (Figs. 5Go and 6Go) or endogenous (Table IIGo) circulating PMN was monitored. Our experimental design ensured that the reduced PMN accumulations observed during LTB4 infusions do not simply reflect the transient drop in circulating PMN induced by i.v. LTB4, as i.d. injections of inflammatory mediators were administered 30 min after starting LTB4 infusion, at which time the neutropenia is fully resolved (32). In addition, changes in skin blood flow could not account for the present results, inasmuch as i.v. infusion of the chemoattractant was found rather to be associated with a transient increase in skin blood flow (37).

In the present study, the inhibition of PMN migration toward various chemoattractants during LTB4 infusion did not result from a heterologous desensitization of circulating PMN to inflammatory mediators; indeed, we have previously shown that blood PMN exposed to i.v. LTB4 selectively lose their ability to respond (neutropenia) to a bolus of LTB4, yet retain their ability to respond to a bolus of FMLP and C5a (32). Furthermore, it has been clearly established previously that LTB4, in contrast to PMN peptidic agonists, does not cause heterologous desensitization (38). This does not exclude the involvement of the LTB4 receptor in the regulation of PMN migration elicited by various chemoattractants but, on the contrary, suggests that LTB4 receptor engagement may be an important common event in PMN transmigration triggered by neutrophil agonists, including IL-8, which is synthesized by endothelial cells at inflammatory sites exposed to IL-1, TNF-{alpha}, or LPS (39, 40).

In other studies, neutrophil agonists such as IL-8 and FMLP were also found to inhibit PMN emigration elicited by a variety of i.d. injected inflammatory mediators when given intravascularly (41, 42). However, the mechanisms underlying the inhibitory effects of LTB4 on PMN extravasation appear to differ from those involved in the similar effects of IL-8 and FMLP. The mechanism of action of IL-8, shared by other peptide chemoattractants including FMLP and C5a, has been suggested to involve cytoskeletal events, more specifically the ability of these agonists to induce rapid polymerization of actin followed by a slower depolymerization phase with concomitant disorganization of focal attachment plaques (42, 43). These events coincide with detachment of tightly adherent PMN from activated endothelial cells in vitro (44), as well as from mesenteric microvessel walls in vivo (42). LTB4 did not cause neutrophil detachment from activated endothelial cells in vitro (44), but rather promoted leukocyte adherence to microvessel walls when applied intravascularly (45, 46). In fact, intravital microscopy observations revealed a prominent and sustained adhesion of PMN exposed to a continuous infusion of LTB4, without evidence for leukocyte emigration or protein leakage in the extravascular compartment (45, 46). This enhanced adhesive interaction of circulating PMN to postcapillary venules during LTB4 infusion appears to be largely mediated by the CD11/CD18 ß2 integrins, because it was almost completely abrogated by the concomitant administration of a CD18-specific mAb (45). Therefore, these observations would suggest that while ß2 integrin-mediated attachment of PMN to ICAM-1 expressed on endothelial cells is a key step in their migration from the systemic microcirculation in response to locally injected chemoattractants and cytokines (47, 48, 49, 50, 51), the ß2 integrin-mediated persistent adhesion of PMN elicited by exposure of circulating PMN to intravascular LTB4 rather hampers emigration by modifying the dynamics of PMN-endothelial cell interactions, e.g., by maintaining a strong persistent adhesion of PMN to the endothelium. Alternatively, as suggested previously by others (37, 42, 46), the presence of elevated concentrations of LTB4 (or other chemoattractants) in the circulation may create a condition at the blood/endothelium interface that hampers the directed migration of PMN through the blood vessel walls. Such interpretations are consistent with the nonselective inhibitory effect of i.v. LTB4 on PMN migration reported herein.

A possible course of events that allows conciliation of our observations supporting a crucial role for 5-LO product formation in PMN in the migration process, as well as the inhibitory effect of intravascular LTB4 on the same process, could be that LTB4, directly generated by PMN (or by endothelial cells through transcellular metabolism of PMN-derived LTA4) upon activation by a chemoattractant, triggers events in endothelial cells (such as retraction and/or changes in the expression of adhesion molecules) (16, 52, 53) that either facilitate emigration or are necessary for the further steps of the transmigration process (Fig. 7Go). LTB4 generated by PMN may also engage PMN LTB4 receptors and further activate the 5-LO pathway in an autocrine manner, resulting in enhanced production of LTA4 and other lipoxygenase products (LTB4, LTC4, and LXA4). In such a scheme of events, it would be expected that ex vivo pretreatment of PMN with a 5-LO product synthesis inhibitor, the direct infusion of drugs that inhibit the synthesis of LTB4 (or blocks its receptors), and the prolonged infusion of LTB4 itself (which causes LTB4 receptor desensitization) each will lead to decreased PMN extravasation independently of the nature of the chemoattractant. In agreement with such a hypothesis, it has been reported that PMN responsitivity to chemoattractants is necessary for the emigration process in vivo (54) and that PMN agonists (C5a, IL-8, FMLP, platelet-activating factor, and LTB4 itself) stimulate LTB4 biosynthesis in PMN (55, 56, 57, 58, 59). Furthermore, ligation of ß2 integrin to its ligand also results in activation of the PMN 5-LO pathway (60). Interestingly, the observations by Pettipher et al. (61), that the systemic (s.c.) administration of 20-hydroxy-LTB4 (which desensitizes LTB4 receptors) or the oral administration of an LTB4 receptor antagonist inhibit both LTB4 and C5a-induced eosinophil accumulation in guinea pig skin, are compatible with the hypothetical scheme of events involving a role for LTB4 receptor engagement in PMN migration elicited by various soluble chemoattractants (Fig. 7Go).



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  		FIGURE 7. Hypothetical scheme of the involvement of 5-LO products and their receptors in the regulation of PMN emigration elicited by chemoattractants, inflammatory cytokines, and LPS. Engagement of PMN chemoattractant receptors on vascular endothelium results in expression of ß2 integrins and firm adhesion of PMN to endothelial cells. PMN chemoattractant receptor engagement also results in activation of the PMN 5-LO pathway and in the biosynthesis of LTB4, independently of the nature of chemoattractant(s) involved, with LPS, TNF-{alpha}, and IL-1 acting indirectly through stimulation of IL-8 formation by endothelial cells. The LTB4 generated may then ligate its receptors either on PMN or endothelial cells and trigger events that are important for transendothelial migration (such as regulation of PMN-endothelium adhesive interaction or possibly endothelial cell retraction). An autocrine amplification loop of 5-LO product synthesis by LTB4 itself (as indicated by the bold arrow) might also be implicated. In this scenario, activation of the 5-LO pathway and ligation of the LTB4 receptor represent common events in the mechanism of PMN emigration elicited by soluble chemoattractants. Consequently, inhibition of 5-LO activity and desensitization (or blockade) of the LTB4 receptors would be expected to down-regulate PMN chemotaxis to all PMN agonists, in agreement with the data reported herein. The scenario also includes the possible involvement of LTC4 generated by endothelial cells (through transcellular metabolism of LTA4) in the regulation of endothelial cell functions (adhesiveness for PMN and retraction) and of LXA4 generated by PMN or platelets (through transcellular metabolism of LTA4) in the regulation of PMN adhesion properties and migration. R, receptor; BLTR, LTB4 receptor; ALXR, LXA4 receptor; CysLTR, cysteinyl LT receptor; cPLA2, cytosolic phospholipase A2; 12-LO, 12-lipoxygenase; 15-LO, 15-lipoxygenase; FLAP, 5-LO activating protein; A4-H, LTA4 hydrolase; C4-S, LTC4 synthase.

 
In conclusion, our data strongly support the concept that 5-LO activation in PMN at the blood/endothelium interface plays an important role in PMN emigration elicited by soluble agonists, supporting the usefulness of drugs interfering with LT biosynthesis for the treatment of inflammatory diseases. In addition, our results also show that elevated levels of intravascular LTB4 potently inhibits PMN emigration, inasmuch as exposure of blood PMN to steady-state elevated concentrations of LTB4 resulted in a dose-dependent inhibition of PMN extravasation. Interestingly, this latter observation may be relevant to pathophysiologic situations associated with elevated plasma levels of LTB4. For example, in ischemia and reperfusion, in which LTB4 has been found to accumulate intravascularly on reperfusion of the ischemic vessels (23, 62), PMN diapedesis is reduced, resulting in delayed neutrophil sequestration in lung and ischemic tissues (23). This may represent a natural antiinflammatory mechanism by which ischemic tissues are protected from the acute inflammatory stress caused by the massive invasion of PMN.


   Aknowledgments
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
References
 
We thank Ms. Tania Lévesque for skillful technical assistance and M. Serge Picard for performing the HPLC analysis of plasma samples. We also thank Dr. David Macari for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by grants from the Medical Research Council of Canada and by the Merck Frosst Center for Therapeutic Research. S.M. was a recipient of a postdoctoral fellowship from the Medical Research Council of Canada, and P.B. and P.E.P. were recipients of scholarships from le Fonds de la Recherche en Santé du Québec, respectively. Back

2 Current address: Faculté de pharmacie, Université de Montréal, C.P. 6128, Succ. Centre-Ville, Montréal, Québec, Canada, H3C 3J7 Back

3 Address correspondence and reprint requests to Dr. Sylvie Marleau, Faculté de pharmacie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, Québec, Canada H3C 3J7. E-mail address: Back

4 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; 5-LO, 5-lipoxygenase; LT, leukotrienes; hr, human recombinant; MPO, myeloperoxidase; PPP, platelet-poor plasma; i.d., intradermal. Back

Received for publication December 11, 1998. Accepted for publication July 7, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Aknowledgments
 References
 

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