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Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia.

Lawrence DW, Bruyninckx WJ, Louis NA, Lublin DM, Stahl GL, Parkos CA, Colgan SP - J. Exp. Med. (2003)

Bottom Line: This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers.Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface.The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, 20 Shattuck St., Boston, MA 02115, USA.

ABSTRACT
Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN-epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70-80-kD, glycosyl-phosphatidyinositol-linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

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Localization of OE-1 antigen to the apical surface. (A) Confocal immunofluorescence was used to define the pattern of OE-1 antigen expression. Shown here is an x-z orientation series demonstrating nearly complete localization to the apical and subapical membrane compartment. (B) An apical confocal section localizing OE-1 (red) with the PMN marker CD11b/18 (clone 44a, green) after PMN transmigration. C represents an x-z orientation series of B. In D, equal amounts of lysates from indicated cell types were resolved on an 10% SDS-PAGE and immunoblotted with OE-1. The OE-1 antigen is expressed in varying amounts in the different cell types. The differences in molecular weights are likely due to differential glycosylation. Epithelial cell lines (KB, T84) express the highest levels of OE-1 antigen, followed by cells normally in close contact with complement proteins (HMVEC, platelets). Primary cell lines such as OKF6 (keratinocytes) express lesser amounts of the OE-1 antigen. (E) Confluent T84 monolayers were biotinylated on the apical (Ap) or basolateral (Bl) surface. Lysates were immunoprecipitated with either an isotype control antibody (C) or OE-1. Proteins were visualized with avidin-HRP to identify only surface, biotinylated DAF (left) or with OE-1 to demonstrate total DAF (right). Lanes labeled “C” represent isotype immunoprecipitation control samples.
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fig2: Localization of OE-1 antigen to the apical surface. (A) Confocal immunofluorescence was used to define the pattern of OE-1 antigen expression. Shown here is an x-z orientation series demonstrating nearly complete localization to the apical and subapical membrane compartment. (B) An apical confocal section localizing OE-1 (red) with the PMN marker CD11b/18 (clone 44a, green) after PMN transmigration. C represents an x-z orientation series of B. In D, equal amounts of lysates from indicated cell types were resolved on an 10% SDS-PAGE and immunoblotted with OE-1. The OE-1 antigen is expressed in varying amounts in the different cell types. The differences in molecular weights are likely due to differential glycosylation. Epithelial cell lines (KB, T84) express the highest levels of OE-1 antigen, followed by cells normally in close contact with complement proteins (HMVEC, platelets). Primary cell lines such as OKF6 (keratinocytes) express lesser amounts of the OE-1 antigen. (E) Confluent T84 monolayers were biotinylated on the apical (Ap) or basolateral (Bl) surface. Lysates were immunoprecipitated with either an isotype control antibody (C) or OE-1. Proteins were visualized with avidin-HRP to identify only surface, biotinylated DAF (left) or with OE-1 to demonstrate total DAF (right). Lanes labeled “C” represent isotype immunoprecipitation control samples.

Mentions: Next, we determined the relative nature and localization of OE-1 antigen. Confocal immunolocalization was used to determine the overall localization on T84 epithelia. As shown in Fig. 2 A, the OE-1 epitope localized exclusively to the apical membrane surface and to subapical membrane domains of polarized cells grown on membrane permeable supports. Localization of the antigen after PMN transmigration in the presence of OE-1 revealed that the OE-1 epitope remains localized to the apical domain and that PMN (localized with anti-CD11b mAb 44a) appear to “cluster” in distinct regions on the apical surface in the presence of OE-1 (Fig. 2, B and C). This clustering of PMN is consistent with previous observations (16), and is likely a result of localized epithelial barrier disruptions, with resultant increases in flux of chemoattractant.


Antiadhesive role of apical decay-accelerating factor (CD55) in human neutrophil transmigration across mucosal epithelia.

Lawrence DW, Bruyninckx WJ, Louis NA, Lublin DM, Stahl GL, Parkos CA, Colgan SP - J. Exp. Med. (2003)

Localization of OE-1 antigen to the apical surface. (A) Confocal immunofluorescence was used to define the pattern of OE-1 antigen expression. Shown here is an x-z orientation series demonstrating nearly complete localization to the apical and subapical membrane compartment. (B) An apical confocal section localizing OE-1 (red) with the PMN marker CD11b/18 (clone 44a, green) after PMN transmigration. C represents an x-z orientation series of B. In D, equal amounts of lysates from indicated cell types were resolved on an 10% SDS-PAGE and immunoblotted with OE-1. The OE-1 antigen is expressed in varying amounts in the different cell types. The differences in molecular weights are likely due to differential glycosylation. Epithelial cell lines (KB, T84) express the highest levels of OE-1 antigen, followed by cells normally in close contact with complement proteins (HMVEC, platelets). Primary cell lines such as OKF6 (keratinocytes) express lesser amounts of the OE-1 antigen. (E) Confluent T84 monolayers were biotinylated on the apical (Ap) or basolateral (Bl) surface. Lysates were immunoprecipitated with either an isotype control antibody (C) or OE-1. Proteins were visualized with avidin-HRP to identify only surface, biotinylated DAF (left) or with OE-1 to demonstrate total DAF (right). Lanes labeled “C” represent isotype immunoprecipitation control samples.
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Related In: Results  -  Collection

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fig2: Localization of OE-1 antigen to the apical surface. (A) Confocal immunofluorescence was used to define the pattern of OE-1 antigen expression. Shown here is an x-z orientation series demonstrating nearly complete localization to the apical and subapical membrane compartment. (B) An apical confocal section localizing OE-1 (red) with the PMN marker CD11b/18 (clone 44a, green) after PMN transmigration. C represents an x-z orientation series of B. In D, equal amounts of lysates from indicated cell types were resolved on an 10% SDS-PAGE and immunoblotted with OE-1. The OE-1 antigen is expressed in varying amounts in the different cell types. The differences in molecular weights are likely due to differential glycosylation. Epithelial cell lines (KB, T84) express the highest levels of OE-1 antigen, followed by cells normally in close contact with complement proteins (HMVEC, platelets). Primary cell lines such as OKF6 (keratinocytes) express lesser amounts of the OE-1 antigen. (E) Confluent T84 monolayers were biotinylated on the apical (Ap) or basolateral (Bl) surface. Lysates were immunoprecipitated with either an isotype control antibody (C) or OE-1. Proteins were visualized with avidin-HRP to identify only surface, biotinylated DAF (left) or with OE-1 to demonstrate total DAF (right). Lanes labeled “C” represent isotype immunoprecipitation control samples.
Mentions: Next, we determined the relative nature and localization of OE-1 antigen. Confocal immunolocalization was used to determine the overall localization on T84 epithelia. As shown in Fig. 2 A, the OE-1 epitope localized exclusively to the apical membrane surface and to subapical membrane domains of polarized cells grown on membrane permeable supports. Localization of the antigen after PMN transmigration in the presence of OE-1 revealed that the OE-1 epitope remains localized to the apical domain and that PMN (localized with anti-CD11b mAb 44a) appear to “cluster” in distinct regions on the apical surface in the presence of OE-1 (Fig. 2, B and C). This clustering of PMN is consistent with previous observations (16), and is likely a result of localized epithelial barrier disruptions, with resultant increases in flux of chemoattractant.

Bottom Line: This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers.Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface.The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

View Article: PubMed Central - PubMed

Affiliation: Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Harvard Medical School, 20 Shattuck St., Boston, MA 02115, USA.

ABSTRACT
Neutrophil migration across mucosal epithelium during inflammatory episodes involves the precise orchestration of a number a cell surface molecules and signaling pathways. After successful migration to the apical epithelial surface, apically localized epithelial proteins may serve to retain PMN at the lumenal surface. At present, identification of apical epithelial ligands and their PMN counter-receptors remain elusive. Therefore, to define the existence of apical epithelial cell surface proteins involved in PMN-epithelial interactions, we screened a panel of antibodies directed against epithelial plasma membranes. This strategy identified one antibody (OE-1) that both localized to the apical cell membrane and significantly inhibited PMN transmigration across epithelial monolayers. Microsequence analysis revealed that OE-1 recognized human decay-accelerating factor (DAF, CD55). DAF is a highly glycosylated, 70-80-kD, glycosyl-phosphatidyinositol-linked protein that functions predominantly as an inhibitor of autologous complement lysis. DAF suppression experiments using antisense oligonucleotides or RNA interference revealed that DAF may function as an antiadhesive molecule promoting the release of PMN from the lumenal surface after transmigration. Similarly, peptides corresponding to the antigen recognition domain of OE-1 resulted in accumulation of PMN on the apical epithelial surface. The elucidation of DAF as an apical epithelial ligand for PMN provides a target for novel anti-inflammatory therapies directed at quelling unwanted inflammatory episodes.

Show MeSH
Related in: MedlinePlus