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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 4, 2004 as doi:10.1096/fj.03-0599fje. |
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* Center for Cardiopulmonary Pharmacology and Department of Pharmacological Sciences, University of Milan, Milan, Italy;
National Jewish Medical and Research Center, Denver, Colorado, USA;
Istituto Neurologico Besta, 20133 Milan, Italy; and
Department of Pathology, University of Verona, Verona, Italy
3Correspondence: Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: angelo.sala{at}unimi.it
SPECIFIC AIMS
The aims of this study were to 1) evaluate the effect of intravascular activation of neutrophils on the formation of cysteinyl leukotrienes (cysLT) and on the permeability of the cerebral microvasculature, 2)identify the cysLT receptor subtype involved in the observed alterations of cerebral vascular permeability, and 3) determine the role of neutrophil 5-lipoxygenase metabolites in neutrophil/cerebral endothelial cell interactions occurring after LPS treatment in vivo in the mouse.
PRINCIPAL FINDINGS
1. Challenge with the chemotactic formylated tripeptide fMLP of neutrophil-perfused brain in vitro resulted in the synthesis of cysLT, accompanied by a significant alteration of endothelial permeability
Analysis of perfusion buffers from isolated guinea pig brain in the presence of purified, fMLP-activated neutrophils showed a significant increase in cys-LT as assessed by specific EIA (40.7±6.7 pg/mL vs. 14.5±4.5 pg/mL in preparations perfused with unstimulated neutrophils, P<0.01). Electrospray ionization tandem mass spectrometry confirmed the results of EIA, unequivocally identifying LTD4 in activated neutrophil perfused preparations only. Mass spectrometric analysis showed the presence of nonenzymatic degradation products of LTA4, a clear index that formation and export of the unstable intermediate in the synthesis of leukotrienes were occurring.
Formation of cysLT was accompanied by alteration of vascular permeability as shown by the statistically significant increase in the wet weight of the preparations compared with preparations perfused with unstimulated neutrophils (Fig. 1
).
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2. Synthesis of cysLT and changes in cerebral vascular permeability associated with the neutrophil activation by fMLP were prevented by inhibition of neutrophilic 5-lipoxygenase
Pretreatment of neutrophils only with a slowly reversible leukotriene synthesis inhibitor (MK-886, 1 µM), followed by washing and infusion into the isolated brain preparations, prevented formation of cysLT (11.5±1.3 pg/mL of cysLT, ns vs. unstimulated neutrophil perfused preparations) and alterations in vascular permeability associated with the activation of neutrophil-perfused isolated brains (Fig. 1)
. Supplementation of the isolated brain preparations with 10 nM of the neutrophilic 5-lipoxygenase metabolite LTB4 was unable to restore the effect observed in the absence of the leukotriene synthesis inhibitor (Fig. 1)
. This agrees with the known activity of cysLTs on vascular permeability and provides evidence that, in cerebral vasculature, biologically relevant concentrations of cysLT may come not only from cells possessing both 5-lipoxygenase and LTC4 synthase (such as mast cells or eosinophils) but also from the cooperation between 5-LO-possessing cells (neutrophils) and cells bearing enzymatic activities able to convert the intermediate LTA4 into LTC4, such as the microsomal glutathione S-transferase 2 (endothelial cells).
3. Activation of the newly described cysLT2 receptor subtype is mainly responsible for the cysLT-dependent alteration of cerebral vascular permeability observed in activated neutrophil perfused brains
Results showed that compound BAY u9773 was more potent than iralukast in preventing an increase in brain wet weight, with an inhibition of >50% at 0.03 µM, whereas a 10-fold higher concentration was required to obtain a significant inhibition using iralukast (Fig. 2
). Increasing the concentration of BAY u9773 (0.3 µM) abolished the protection and indeed worsened the inflammatory reaction (Fig. 2)
, in agreement with a potential partial agonist activity of the compound.
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4. Inhibition of neutrophil leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling
Quantitative evaluation of neutrophil interactions with the cerebral vascular endothelium was carried out using a model of intravital microscopy in LPS-treated mice. Fluorescence-labeled rolling neutrophils constituted a small (13.2±4.73%, n=7) but clearly identifiable fraction of the total cell number, which was not influenced by the hemodynamic parameters. Treatment of neutrophils before infusion with an inhibitor of leukotriene synthesis (MK-886) did not alter the distribution of rolling leukocytes within different classes of velocity or the total fraction of rolling neutrophils. On the contrary, the percentage of firmly adhering cells over the total number of flowing cells was significantly reduced from 8.4 ± 3.2 to 4.7 ± 2.1 (n=7) by inhibition of neutrophilic 5-LO enzyme activity, suggesting a contribution of neutrophilic 5-LO metabolites to the process leading to prolonged neutrophil/endothelial cell interactions.
CONCLUSIONS
The current study provided evidence that challenge of neutrophils within an intact cerebral microvascular bed in vitro causes the formation of cysLT, associated with the development of significant alterations of vascular permeability. Results obtained from pretreating neutrophils with the 5-LO inhibitor MK886 indicate that intact 5-LO activity in neutrophils is required for the synthesis of cysLT during neutrophil perfusion of isolated brains. This strongly supports the hypothesis that the observed formation of cysLT is the result of transcellular biosynthesis whereby neutrophil-derived leukotriene A4 (LTA4) is handed over to endothelial cells and converted into cysLT. When primed with GM-CSF and activated with fMLP, neutrophils alone synthesized LTB4 and its 
-oxidized metabolites, but no cysLT was detected. LC/MS/MS analysis provided evidence for the presence of nonenzymatic degradation products of the unstable allelic epoxide LTA4, namely, 
6-trans-LTB4 isomers, confirming that a significant quota of the LTA4 synthesized upon receptor-mediated activation of neutrophils is released extracellularly for potential uptake and metabolism by adhering cells.
Inhibition of 5-LO in neutrophils not only caused complete suppression of fMLP-induced formation of cysLT but prevented the development of cerebral edema, suggesting a causal relationship between transcellular synthesis of cysLT and the changes in vascular permeability.
We next addressed the issue of which receptor subtype could mediate the effect on vascular permeability. It is known that cysLT acts via two classes of G-protein coupled receptors, denoted cysLT1R and cysLT2R. In addition of being the only known compound able to exert antagonistic activity on cysLT2 receptor, BAY u9773 has been reported to be an agonist on the same receptor. In our model, a potential overlapping of agonistic and antagonistic activity is indicated by the fact that while significantly effective at 0.03 µM, BAY u9773 is only slightly more effective at 0.1 µM and loses its activity at the highest concentration tested (0.3 µM). Although affecting the edema formation induced by neutrophil activation, BAY u9773 does not reach the same protection (
100%) afforded by the pretreatment with the leukotriene synthesis inhibitor MK886. Iralukast is able to partially protect against the alteration of vascular permeability induced by neutrophil activation suggesting that cysLT1 receptor can also be involved. The concentrations necessary for this activity appeared to be unrelated to the affinity of iralukast for bronchial cysLT1 receptor as we have previously reported.
To further evaluate the contribution of 5-LO metabolites to the complex interplay between circulating cells and vascular walls, we extended our investigation to an in vivo model of cerebral vessel intravital microscopy. The effect seen on firm adhesion suggests that arachidonic acid metabolites elaborated by 5-LO in neutrophils may also play a role in their juxtaposition onto the intima of cerebral vasculature. In this case, neutrophil-derived LTB4 may represent the most likely candidate since it may stimulate ß2 integrin-dependent adhesion of neutrophils. On the other hand, neutrophil/endothelial cell transcellular interactions may grant formation of cysLT within endothelial cells, where they could act as intracrine agonist for PAF formation and strengthen neutrophil binding or directly activate the neutrophil via the cysLT1 receptor. This may bring a full cycle to closure, as it may represent a positive feedback whereby transcellular synthesis of cysLT will enhance adhesion and prompt additional formation via neutrophil/endothelial cell cooperation.
As evidence points to a pivotal role for white cell adhesion to vessel wall in driving brain damage, the results obtained in this study strongly propose the crosstalk of arachidonic acid metabolites at the neutrophil/endothelial cells interface as an important physiopathologic mechanism during cerebral inflammatory reactions. They provide the first evidence supporting the involvement of the novel cysLT2R in the known effects of cysLT on vascular permeability.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0599fje; 
2 A.D.G. and C.C. contributed equally to the present work.
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