Published online before print May 8, 2003
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Departments of
* Clinical and Experimental Medicine and Pharmacology and
Department of Experimental Pharmacology, University "Federico II", Napoli, Italy; and
Biomorphology, School of Medicine, University of Messina Torre Biologica, Policlinico Universitario, Italy
Correspondence: Salvatore Cuzzocrea, Ph.D., Institute of Pharmacology, School of Medicine, University of Messina, via C. Valeria, Torre Biologica, Policlinico Universitario, 98123 Messina Italy. E-mail: salvator{at}unime.it
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Key Words: adhesion molecules • inflammation
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The conversion of AA to LT A4 (LTA4) by the enzyme 5-LO is the first step in the synthesis of all LT. LTA4 can be converted to LTB4 by LTA4 hydrolase or conjugated with reduced glutathione by LTC4 synthase to form LTC4.
The biologic activities of LT suggest that they are mediators of acute inflammatory and immediate hypersensitivity responses. Peptidyl LT, which are released by leukocytes in response to inflammatory and immunologic stimuli, cause contraction of endothelial cells, resulting in increased permeability of postcapillary venules [2 ]. The peptidyl LT are also potent bronchoconstrictors [3 ] and continue to be considered important mediators of allergic disease of the airways [4 , 5 ]. Therefore, the peptidyl LT and LTB4 increase the adhesion of leukocytes to endothelial cells [2 , 6 ].
In the last few years, various studies have gained substantial insight into the importance of specific adhesion molecules and mediators in the following processes, which finally result in the recruitment of polymorphonuclear leukocytes (PMNs) at a specific site of inflammation: margination, capture, and rolling on the vascular endothelium; activation and firm adhesion; diapedesis through gaps between endothelial cells in postcapillary venules; and finally, migration along a gradient of chemokines. Activated PMNs, therefore, play a crucial role in the destruction of foreign antigens and the breakdown and remodeling of injured tissue. Leukocyte-endothelial interactions involve a complex interplay among adhesion glycoproteins [i.e., integrins, members of the immunoglobulin (Ig) superfamily, and selectins]. One member of the selectin family, P-selectin, is rapidly translocated from the Weibel–Palade bodies to the endothelial cell surface upon activation of endothelial cells with thrombin, histamine, hypoxia-reoxygenation, or oxygen-derived free radicals [7 , 8 ].
P-selectin promotes rolling of leukocytes on the endothelium. E-selectin is expressed on inflamed endothelial cells in response to treatment with inflammatory cytokines [9 ]. Intravital microscopic experiments have shown that its function in mediating leukocyte rolling is largely redundant with that of P-selectin [10 11 12 ]. Consequently, E-selectin-deficient mice have only a subtle defect in leukocyte rolling, as shown by much faster rolling velocities in these mice [12 ]. In addition to mediating leukocyte rolling, E-selectin participates in the conversion of rolling to firm adhesion. E-selectin-deficient mice have a reduced number of firmly adherent leukocytes in response to local chemoattractant [13 ] or cytokine stimulation [14 ]. This defect may be related to the more rapid rolling velocities in the absence of E-selectin, which is expressed in skin microvessels under baseline conditions [15 ]. There is also some evidence that E-selectin is of particular importance in skin inflammation, as it supports the recruitment of skin-specific T lymphocytes [16 ]. The rolling of leukocytes is the first step in the interactions of leukocytes with the endothelium and facilitates the activation and adherence of PMNs [7 , 17 ]. The firm adhesion of PMNs to the endothelium, however, is a complex phenomenon, which also involves other endothelium-based adhesion molecules. In fact, endothelial-adhesion molecules are considered to play a pivotal role in the localization and development of an inflammatory reaction [18 ].
Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule normally expressed at a low basal level, but various inflammatory mediators such as interleukin-1 and tumor necrosis factor 
can enhance its expression [19
]. Vascular cell adhesion molecule-1 (VCAM-1) contains six or seven Ig domains and is expressed on large and small vessels only after the endothelial cells are stimulated by cytokines. Primarily, VCAM-1 is an endothelial ligand for very late antigen-1 or 4ß1 of the ß1 subfamily of integrins and for integrin 4ß7. VCAM-1 promotes the adhesion of lymphocytes, monocytes, eosinophils, and basophils. It is interesting that certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites.
Injection of carrageenan into the pleural space leads to pleurisy, PMN infiltration, and lung injury. Models of carrageenan-induced pleurisy have been widely used to investigate the pathophysiology of acute inflammation and also to evaluate the efficacy of drugs in inflammation. It is interesting that 5-LO knockout mice (5-LOKO) have not been used to elucidate whether 5-LO LT plays a role in the PMN infiltration in this model. This is surprising, as the injection of carrageenan leads to a rapid and substantial rise in LT [20 ]. In this study, we have investigated the role of 5-LO in a model of carrageenan-induced pleurisy using 5-LOKO mice. To characterize the role of 5-LO in this model of acute inflammation, we have determined the following endpoints of the inflammatory response in 5-LOKO mice: exudate formation, PMN infiltration, expression of adhesion molecules (ICAM-1, VCAM-1, P-selectin, E-selectin), and lung injury.
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Carrageenan-induced pleurisy
Mice were anaesthetized with isoflurane and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.1 ml) or saline containing 2% 
-carrageenan (0.1 ml) was injected into the pleural cavity. The skin incision was closed with a suture, and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO2. The chest was carefully opened, and the pleural cavity was rinsed with 1 ml saline solution containing heparin (5 U/ml) and indomethacin (10 µg/ml). The exudate and washing solution were removed by aspiration, and the total volume was measured. Any exudate that was contaminated with blood was discarded. The amount of exudate was calculated by subtracting the volume injected (1 ml) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate-buffered saline (PBS) and counted with an optical microscope in a Burker’s chamber after vital trypan blue staining.
Measurement of nitrite/nitrate
Nitrite/nitrate (NOx) production, an indicator of nitric oxide (NO) synthesis, was measured in pleural exudate. At first, the nitrate in the supernatant was incubated with nitrate reductase (0.1 U/ml) and reduced nicotinamide adenine dinucleotide phosphate (1 mM) and flavin adenine dinucleotide (50 µM) at 37°C for 15 min. Then, another incubation with lactic dehydrogenase (100 U/ml) and sodium pyruvate (10 mM) followed for 5 min. The nitrite concentration in the samples was measured by the Griess reaction, by adding 100 µl Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H2O and 1% sulfanilamide in 5% concentrated H2PO4; vol. 1:1) to 100-µl samples. The optical density at 550 nm (OD550) was measured using enzyme-linked immunosorbent assay (ELISA) microplate reader (SLT-Labinstruments, Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution. Levels of NOx are expressed as ng rat-1.
Immunohistochemical localization of E-selectin, P-Selectin, VCAM-1, and ICAM-1
At 4 h after carrageenan administration, the lungs were fixed in 10% buffered formaldehyde, and 8-µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating the section in 2% normal goat serum in PBS for 20 min. Endogenous biotin- or avidin-binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with primary anti-E-selectin antibody (1:1000), anti-VCAM antibody (1:500), anti-P-selectin antibody (1:500), anti-ICAM-1 antibody (1:500), anti-myeloperoxidase (MPO) antibody (1:100), or control solutions. Controls included buffer alone or nonspecific, purified rabbit IgG. Immunocytochemistry photographs (n=5) were assessed by densitometry. By using Optilab Graftek software on a Macintosh personal computer (CPU G3-266), the assay was performed.
Light microscopy
Lung biopsies were taken at 4 h after injection of carrageenan. The biopsies were fixed for 1 week in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated by graded ethanol, and embedded in Paraplast (Sherwood Medical, Mahwah, NJ). Tissue sections (7-µm thickness) were deparaffinized with xylene, stained with haematoxylin/eosin, and studied using light microscopy (Dialux 22 Leitz).
Determination of MPO activity
MPO activity, an indicator of PMN accumulation, was determined as described previously [21
]. At 4 h after intrapleural injection of carrageenan, lung tissues were obtained and weighed. Each piece of tissue was homogenized in a solution containing 0.5% hexadecyltrimethyl ammonium bromide dissolved in 10 mM potassium-phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetramethylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 µmol peroxide min at 37°C and was expressed in mU/g of wet tissue.
Materials
Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Co. (Milan, Italy). Primary monoclonal P-selectin (CD62P) or ICAM-1 (CD54) for immunohistochemistry was purchased by PharMingen (San Diego, CA). The primary monoclonal antibodies directed at P-selectin or ICAM-1 for ELISA were obtained from R & D Systems (Minneapolis, MN). Reagents and secondary and nonspecific IgG antibody for immunohistochemical analysis were from Vector Laboratories (Burlingame, CA). Primary monoclonal antipoly (adenosine 5'-diphosphate-ribose) antibody was purchased by Alexis (San Diego, CA). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare, Thetford, Norfolk, UK).
Data analysis
All values in the figures and text are expressed as mean ± SEM of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A Pvalue less than 0.05 was considered significant.
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Figure 1. (a) Exudate volume; (b) leukocyte accumulation. 5-LOKO mice show a significant decrease of pleural exudate and leukocyte migration. Responses in 5-LOWT controls and 5-LOKO animals were compared. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus vehicle.  , P < 0.01, versus 5-LOWT mice.
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Figure 2. Representative lung sections from a carrageenan-treated 5-LOWT demonstrate inflammatory infiltration by neutrophil (A). Lung sections from carrageenan-treated 5-LOKO mice (B) demonstrate reduced, inflammatory infiltration. Original magnification, 125x. Figure
is representative of at least three experiments performed on different experimental days.
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Figure 3. MPO activity in the lungs of carrageenan-treated mice killed at 4 h. MPO activity was significantly increased in the lungs of the carrageenan-treated 5-LOWT mice in comparison with sham mice. 5-LOKO mice show a significant reduction of the carrageenan-induced increase in MPO activity. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus vehicle. , P < 0.01, versus 5-LOWT mice.
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Figure 4. Immunohistochemical localization of MPO. Staining of lung tissue sections obtained from sham-operated rats with anti-MPO antibody showed a nonspecific staining along bronchial epithelium (A). Four hours following carrageenan injection, MPO immunoreactivity was significantly present in the lungs from 5-LOWT mice (B and B1). In the lungs of the carrageenan-treated 5-LOKO (C), no positive staining was observed. Original magnification, 375x. Figure is representative of at least three experiments performed on different experimental days.
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Figure 5. Immunohistochemical localization of ICAM-1. Staining of lung tissue sections obtained from sham-operated rats with anti-ICAM-1 antibody showed a specific staining along the bronchial epithelium, demonstrating that ICAM-1 is constitutively expressed (A). Four hours following carrageenan injection, ICAM-1 immunoreactivity was significantly expressed more in the lungs from 5-LOWT mice (B). In the lungs of the carrageenan-treated 5-LOKO (C), significantly less positive staining was found. Original magnification, 188x. Figure is representative of at least three experiments performed on different experimental days.
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Figure 6. Immunohistochemical localization of P-selectin. Staining was absent in control tissue (A). Four hours following carrageenan injection, P-selectin immunoreactivity was present in the lungs from 5-LOWT mice (B). In the lungs of the carrageenan-treated 5-LOKO (C), significantly less positive staining was found. Original magnification, 188x. Figure is representative of at least three experiments performed on different experimental days.
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Figure 7. Immunohistochemical localization of E-selectin. Staining was absent in control tissue (A). Four hours following carrageenan injection, E-selectin immunoreactivity was present in the lungs from 5-LOWT mice (B). In the lungs of the carrageenan-treated 5-LOKO (C), significantly less positive staining was found. Original magnification, 188x. Figure is representative of at least three experiments performed on different experimental days.
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Figure 8. Immunohistochemical localization of VCAM-1. Staining was absent in control tissue (A). Four hours following carrageenan injection, VCAM-1 immunoreactivity was present in the lungs from 5-LOWT mice (B). In the lungs of the carrageenan-treated 5-LOKO (C), significantly less positive staining was found. Original magnification, 188x. Figure is representative of at least three experiments performed on different experimental days.
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Figure 9. Nitrite and nitrate exudate levels at 4 h after carrageenan administration. Nitrite and nitrate exudate levels in carrageenan-treated 5-LOWT mice were significantly increased versus the sham group. The genetic alteration of the 5-LO gene did not affect the increase of the levels of Nox. Data are means ± SEM of 10 mice for each group. *, P < 0.01, versus vehicle.
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Using mice in which the gene for 5-LO was deleted (5-LOKO mice), we demonstrate here that the activation of 5-LO mediates leukocyte-endothelial interactions by regulating the expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 during acute inflammation. In 5-LOKO mice subjected to carrageenan-induced pleurisy, the up-regulation of P-selectin, E-selectin, ICAM-1, and VCAM-1 in the lung was largely attenuated. Endothelial cells appear to be major regulators of the neutrophil traffic, regulating the process of neutrophil chemoattraction, adhesion, and emigration from the vasculature to the tissue. P-selectin is rapidly recruited to the cell surface of platelets or endothelial cells from performed storage pools after exposure to, e.g., hydrogen peroxide, thrombin, histamine, or complement and allows the leukocytes to roll along the endothelium [29 30 31 ]. E-selectin is expressed in skin microvessels under baseline conditions [15 ], and there is some evidence that E-selectin is of particular importance in skin inflammation, as it supports the recruitment of skin-specific T lymphocytes [16 ]. ICAM-1 is constitutively expressed on the surface of endothelial cells and is then involved in the neutrophil adhesion [32 ]. Hypoxic or injured endothelial cells synthesize proinflammatory cytokines, which can up-regulate endothelial expression of the constitutive adhesion molecule ICAM-1 in an autocrine manner [33 , 34 ]. Significant expression of ICAM-1 in microvessels of previously ischemic tissues occurs within 1 h after reperfusion [35 , 36 ]. VCAM-1 is expressed on large and small vessels only after the endothelial cells are stimulated by cytokines. Recently, it has been demonstrated that certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites. The up-regulation expression of P-selectin, E-selectin, ICAM-1, and VCAM-1 corresponds with the induction of neutrophil recruitment, which is maximal within the first hour of reperfusion and persists, at a lower rate, in the late phase of reperfusion [37 , 38 ]. In accordance with these findings, we observed that carrageenan (within 4 h) induced the appearance of P-selectin on the endothelial vascular wall and up-regulated the surface expression of E-selectin, ICAM-1, and VCAM-1 on endothelial cells in the lung section from 5-LOWT mice. The genetic inhibition of 5-LOKO mice abolished the expression of P-selectin and the up-regulation of E-selectin, ICAM-1, and VCAM-1 (Figs. 4 5 6 7) but did not affect the constitutive expression of E-selectin, ICAM-1, and VCAM-1 on endothelial cells (data not shown). These results suggest that inhibition of 5-LO activity may interfere with the interaction of neutrophils and endothelial cells at the early rolling phase mediated by P-selectin and E-selectin and at the late firm adhesion phase mediated by ICAM-1 and VCAM-1. The absence of an increased expression of the adhesion molecules in the lung tissue of carrageenan-treated 5-LOKO mice correlated with the reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPOs and with the moderation of the tissue damage as evaluated by histological examination. It is noteworthy, however, that tissue MPO activity was not completely abolished. This result is consistent with previous studies demonstrating that constitutive levels of ICAM-1 appear to be sufficient to support a lower degree of CD11/CD18-dependent, transendothelial migration of activated neutrophils [39 , 40 ].
In conclusion, the data presented here demonstrate that 5-LO is involved in the regulation of the expression of adhesion molecules and that consequently, 5-LO plays a role in the tissue infiltration of neutrophils. Taken together, the data presented in the present study and in another recent report [41 ] demonstrate that 5-LO regulates the infiltration of neutrophils into the inflamed tissues via a number of distinct mechanisms. The discovery of the concept that 5-LO regulates neutrophil trafficking may provide new insights in the interpretation of recent reports demonstrating the protective effect of 5-LO inhibition in experimental models of ischemia-reperfusion injury and inflammation. In our experimental model, we could not detect any significant interaction between 5-LO and NO pathways, as the deletion of the 5-LO gene did not affect NOx production. These results are in agreement with other observations. In fact, in RAW 264.7 and J774 macrophages, LT do not regulate NO generation [42 , 43 ].
Thus, we propose the following positive feedback cycle in acute lung inflammation: early reactive oxygen species (ROS) production >> 5-LO >> endothelial injury >> adhesion molecule expression >> PMN infiltration >> more ROS production >> tissues damage. Inhibition of 5-LO would interrupt this cycle at the level of endothelial injury, preventing the neutrophil infiltration.
Based on the results of the present study and on the important role of neutrophil infiltration in lung inflammation, further investigations, using LT receptor antagonists or inhibitors of 5-LO, will be performed to clarify the exact mechanism underlying the observed anti-inflammatory events.
Received October 2, 2002; revised February 11, 2003; accepted February 14, 2003.
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