shown in studies using MAbs(21) and several polysaccharides (22). L-selectin is constitutively
expressed on the cell surface of neutrophils, and signal transductionthrough chemoattrac-
tant receptors results in rapid activationof L-selectin by phosphorylation (23), followed by proteolyticcleavage from the neutrophil cell surface (24). Activation ofL-selectin increases its binding activity, enabling initialcontact with endothelial mucin-like carbohydrate ligands
(25).Furthermore, recent studies showed that phosphorylation of L-selectinafter chemoat-
tractant activation occurs in conjunction withthe dissociation of calmodulin from the cyto- plasmatic domainof L-selectin before proteolysis from the neutrophil cell surface(26,27). In this study, isoflurane inhibited the chemoattractant-induceddownregulation of L-selectin. Spontaneous shedding of L-selectinfrom unstimulated leukocytes (26), which is mediated by
a membrane-associatedL-selectin sheddase, could be inhibited by metalloproteinaseinhibi-
tors (24). Accordingly, a direct effect of isofluraneon the proteolytic sheddase seems unlikely, because L-selectinexpression was not altered on unstimulated neutrophils. Therefore,we suggest that inhibition of the chemo-attractant-induced downregulationof L-selectin by isoflurane might be due to a reduced activationof L-selectin. Because activation of L-selectin enables initialcontact to endothelial cells, our findings may provide a furthermechanism of the isoflurane-induced inhibition of neutrophiladhesion to endothelial cells in ischemia- reperfusion injury.
The leukocyte β2-integrins CD11a and CD11b are involvedin numerous aspects of leuko-
cyte function, including tight adhesionto endothelial cells, transmigration phagocytosis, and neutrophilactivation (15). Patients lacking these integrins are susceptibleto severe infections (28), but excessive activation contributesto sustained inflammation, reperfusion injury, and tissue damage(1,2). The results of this study confirmed those from Möbertet al. (12), who
suggested that inhibition of the upregulationof CD11b is one relevant mechanism respon-
sible for the reducedadhesion of neutrophils to endothelial cells. As a possibleunderlying mechanism, the authors speculated that the volatileanesthetics might have entered the plasma membrane, resultingin membrane expansion and thereby decreasing the upward regulationof CD11b. In this study we used two different chemoattractantsto gain further
insight into the underlying mechanism of theisoflurane-induced inhibition of CD11b. The
ability of PMA,a direct activator of PKC, to induce activation of CD11b notinhibited by
isoflurane supports the conclusion that plasmamembrane expansion is unlikely to be the
underlying mechanism.
Engagement of FMLP at the G protein-linked receptor leads tothe activation of phospho-
lipase C and D, generation of inositoltriphosphate, activation of PKC, and calcium influx (29).
However,signaling events downstream of PKC leading to CD11b activationare only incom-
pletely established but seem to involve the activationof Src family kinases and mitogen-
activated protein kinase p38(30). However, two investigations showed that FMLP-induced
p38 (30,31), suggesting an alternative signalingpathway. Because isoflurane did not affect PMA-induced activationof CD11b, we speculate that isoflurane may alter either thesignaling
pathway upstream of PKC or the PKC-independent signalingpathway. However, further stud-
ies are required to identify theeffect of isoflurane on chemoattractant-induced neutrophil signalingpathways.
CD11a is expressed on neutrophils and mediates interactionsof neutrophils with the endothelium and transendothelial migrationvia binding to endothelial ICAM-1 (32,33). After activationwith either FMLP or PMA, CD11a is redistributed over the cellmembrane, thus forming high-avidity clusters and inducing ligandbinding activity (34). In this study, stimu-
lation with eitherFMLP or PMA resulted in increased CD11a MFI on the neutrophilsurface,
representing activation of the CD11a ligand bindingactivity. Because isoflurane reduced the
mean CD11a fluorescenceintensity in the unstimulated samples and after stimulationwith
FMLP, it is possible that isoflurane may inhibit the bindingof CD11a to endothelial ICAM-1. We suggest that the inhibitionof CD11a ligand binding activity might be another reason for the reduced adhesion of neutrophils to endothelial cells inthe presence of isoflurane.
In conclusion, the results of this study indicate that the inhibitingeffect of isoflurane on
neutrophil recruitment may be mediatedby a decreased activation of L-selectin and by at-
tenuation ofactivation of the β2-integrins CD11a and CD11b onthe neutrophil surface.
Acknowledgments
Supported by START, a research grant of the Rheinisch-WestfälischeTechnische Hochschule
4
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