Eosinophils (EOS) and neutrophils (PMN) display different patterns of accumulation during various inflammatory reactions. We hypothesized that EOS and PMN may differ in their ligands for P-selectin, and that these ligands may differ from those previously identified for E-selectin. Recombinant human P-selectin was immobilized on plastic surfaces and adhesion of 51Cr-labeled human EOS or PMN was compared. EOS and PMN adhered in a concentration-dependent fashion, with similar maximal net adhesion. Preincubation with a blocking P-selectin antibody inhibited adhesion of both cell types, whereas a non-blocking antibody did not. To determine if the counterligands were sialylated proteins, cells were treated with various glycosidases and proteases before testing adhesion. Neuraminidase treatment markedly inhibited binding of both cell types, while endo-beta-galactosidase had no significant effect. Pretreatment with several proteases reduced adhesion of both cell types, although they consistently caused a greater inhibition of PMN binding than EOS binding. To determine whether the P-selectin ligands were surface structures whose expression or function may be altered by cell activation, leukocytes were pretreated with various stimuli; only platelet-activating factor (PAF) treatment reduced the capacity of leukocytes to adhere to P-selectin. Thus, the counterligands for P-selectin on EOS and PMN are similar sialylated, protease-sensitive, endo-beta-galactosidase-resistant structures, whose function and/or expression is reduced following treatment with PAF. These characteristics are clearly different than those reported for EOS and PMN ligands for E-selectin, and suggest disparate roles for P-selectin and E-selectin during EOS and PMN recruitment during inflammatory responses in vivo.
Both neutrophils and eosinophils have been shown to bind to the inducible endothelial cell adhesion molecule E-selectin. For neutrophils, one of the reported ligands for E-selectin is the sialylated Lewis X Ag (sLe(x)). To analyze the counterligands on eosinophils for E-selectin, adhesion assays were performed in which purified leukocytes were allowed to adhere to a soluble recombinant form of the molecule immobilized on plastic plates. Eosinophils, like neutrophils, bound to immobilized E-selectin, but significantly more neutrophils than eosinophils adhered in this assay. Consistent with the greater ability of neutrophils to bind E-selectin was the observation by flow cytometry that neutrophils expressed significant levels of sLe(x) and a sialylated dimeric form of the Le(x) Ag (sialyl-dimeric Le(x), or sialyl-stage-specific embryonic Ag-1, recognized by mAb FH6), whereas the expression of these epitopes on eosinophils was extremely low or undetectable. Expression was similar on eosinophils from allergic and nonallergic donors, and was not altered on eosinophils after induction of L-selectin shedding in vitro by treatment with platelet-activating factor. For both eosinophils and neutrophils, treatment with sialidase was associated with the complete elimination of sLe(x) and sialyl-dimeric Le(x) surface expression, and abolished leukocyte adhesion to E-selectin. Another glycosidase, endo-beta-galactosidase, which specifically cleaves the beta 1-4 galactose linkage to N-acetyl-glucosamine when it exists in an extended chain form such as that found in sialyl-dimeric Le(x), significantly inhibited eosinophil and neutrophil adhesion and expression of sialyl-dimeric Le(x). Such treatment also reduced sLe(x) expression on eosinophils, while having little effect on total neutrophil sLe(x) expression. For both eosinophils and neutrophils the sialylated ligand did not appear to be a glycoprotein because pretreatment of leukocytes with several proteases had no effect on adhesion to E-selectin or on expression of sLe(x) and sialyl-dimeric Le(x). These data suggest that eosinophils, like neutrophils, use sialylated, protease-resistant structures to bind to E-selectin, although the eosinophil expresses much lower levels of these structures on its surface. A major proportion of the sLe(x)-containing E-selectin ligand on the surface of eosinophils appears to be in the form of sialyl-dimeric Le(x), whereas this represents a minor proportion on the surface of neutrophils. Based on results using endo-beta-galactosidase, it appears that these cells may rely disproportionately upon the cell surface sialyl-dimeric Le(x) to bind to E-selectin.(ABSTRACT TRUNCATED AT 400 WORDS)
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.