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The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells.

Maddaluno L, Verbrugge SE, Martinoli C, Matteoli G, Chiavelli A, Zeng Y, Williams ED, Rescigno M, Cavallaro U - J. Exp. Med. (2009)

Bottom Line: In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo.Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration.Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs.

View Article: PubMed Central - PubMed

Affiliation: The FIRC Institute of Molecular Oncology, 20139 Milan, Italy.

ABSTRACT
The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.

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TNF-α induces L1 expression in endothelium. (A)HUVEC or 1G11 cells were starved of serum and endothelial growth factorsand then treated with 20 ng/ml TNF-α for 3 h, followed by FACSanalysis for L1 expression. (B) HUVEC were treated with 20 ng/mlTNF-α for the indicated time lengths, followed by FACSanalysis for L1 expression. The data refer to the percentage ofL1-positive cells in a representative experiment. Each experiment wasrepeated three times with similar results. (C) HUVECs were treated with20 ng/ml TNF-α for the indicated time lengths before isolationof RNA and quantitative RT-PCR analysis for L1 expression. Datarepresent the means ± SEM of three experiments performed.*, P < 0.05 (relative to untreated cells).(D–I) C57BL/6 mice (three mice per group) were subjected tosubcutaneous injection of 100 µl of either vehicle(D–F) or 40 ng/ml TNF-α (G-I) and sacrificed after16 h. Skin fragments from the injection sites were fixed and costainedfor PECAM-1 (red) and L1 (green) before confocal analysis. Insets show ablood vessel cross section with the ECs positive for both PECAM-1 andL1. The arrow in F indicates an L1-positive nerve that served as aninternal control. Bars, 40 µm.
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fig7: TNF-α induces L1 expression in endothelium. (A)HUVEC or 1G11 cells were starved of serum and endothelial growth factorsand then treated with 20 ng/ml TNF-α for 3 h, followed by FACSanalysis for L1 expression. (B) HUVEC were treated with 20 ng/mlTNF-α for the indicated time lengths, followed by FACSanalysis for L1 expression. The data refer to the percentage ofL1-positive cells in a representative experiment. Each experiment wasrepeated three times with similar results. (C) HUVECs were treated with20 ng/ml TNF-α for the indicated time lengths before isolationof RNA and quantitative RT-PCR analysis for L1 expression. Datarepresent the means ± SEM of three experiments performed.*, P < 0.05 (relative to untreated cells).(D–I) C57BL/6 mice (three mice per group) were subjected tosubcutaneous injection of 100 µl of either vehicle(D–F) or 40 ng/ml TNF-α (G-I) and sacrificed after16 h. Skin fragments from the injection sites were fixed and costainedfor PECAM-1 (red) and L1 (green) before confocal analysis. Insets show ablood vessel cross section with the ECs positive for both PECAM-1 andL1. The arrow in F indicates an L1-positive nerve that served as aninternal control. Bars, 40 µm.

Mentions: Given the difference between human and murine immune systems, we asked whether L1is also involved in the transendothelial migration of human DCs. To this goal,we used human monocyte-derived DCs (moDCs), which express moderate levels of L1(Fig. 5 A; reference 5). The maturation of human moDCs was notaccompanied by changes in L1 levels (not depicted), confirming our observationson mouse bone marrow–derived DCs (Fig. S3 B). To evaluate the role ofL1 in transendothelial migration, CFSE-labeled moDCs were pretreated with CE7, amonoclonal antibody that has been previously shown to neutralize L1 function(21), and then allowed to cross amonolayer of TNF-α–activated human umbilical vein ECs(HUVECs). The inactivation of L1 in moDCs with CE7 resulted in a dramaticreduction of the transendothelial migration as compared with moDCs treated withan irrelevant antibody (Fig. 5 B). Giventhe expression of L1 in activated ECs (10) as well as in TNF-α–treated HUVECs (see Fig. 7 A), we also assessed thecontribution of vascular L1 to DC transendothelial migration by pretreatingHUVECs with CE7 before transmigration assays. The inactivation of endothelial L1caused a reduction in the transmigratory activity of moDCs (Fig. 5 B). Finally, when L1 was neutralized in both DCsand HUVECs, no additive effect was observed as compared with the inactivation inthe individual cell types (Fig. 5 B).Notably, CE7 had no effect on chemokine-induced migration of moDCs (notdepicted), which is in line with the results on L1-deficient mouse DCs (Fig. S4B). Thus, L1 function is required for the trafficking of human DCs through anendothelial barrier.


The adhesion molecule L1 regulates transendothelial migration and trafficking of dendritic cells.

Maddaluno L, Verbrugge SE, Martinoli C, Matteoli G, Chiavelli A, Zeng Y, Williams ED, Rescigno M, Cavallaro U - J. Exp. Med. (2009)

TNF-α induces L1 expression in endothelium. (A)HUVEC or 1G11 cells were starved of serum and endothelial growth factorsand then treated with 20 ng/ml TNF-α for 3 h, followed by FACSanalysis for L1 expression. (B) HUVEC were treated with 20 ng/mlTNF-α for the indicated time lengths, followed by FACSanalysis for L1 expression. The data refer to the percentage ofL1-positive cells in a representative experiment. Each experiment wasrepeated three times with similar results. (C) HUVECs were treated with20 ng/ml TNF-α for the indicated time lengths before isolationof RNA and quantitative RT-PCR analysis for L1 expression. Datarepresent the means ± SEM of three experiments performed.*, P < 0.05 (relative to untreated cells).(D–I) C57BL/6 mice (three mice per group) were subjected tosubcutaneous injection of 100 µl of either vehicle(D–F) or 40 ng/ml TNF-α (G-I) and sacrificed after16 h. Skin fragments from the injection sites were fixed and costainedfor PECAM-1 (red) and L1 (green) before confocal analysis. Insets show ablood vessel cross section with the ECs positive for both PECAM-1 andL1. The arrow in F indicates an L1-positive nerve that served as aninternal control. Bars, 40 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2664975&req=5

fig7: TNF-α induces L1 expression in endothelium. (A)HUVEC or 1G11 cells were starved of serum and endothelial growth factorsand then treated with 20 ng/ml TNF-α for 3 h, followed by FACSanalysis for L1 expression. (B) HUVEC were treated with 20 ng/mlTNF-α for the indicated time lengths, followed by FACSanalysis for L1 expression. The data refer to the percentage ofL1-positive cells in a representative experiment. Each experiment wasrepeated three times with similar results. (C) HUVECs were treated with20 ng/ml TNF-α for the indicated time lengths before isolationof RNA and quantitative RT-PCR analysis for L1 expression. Datarepresent the means ± SEM of three experiments performed.*, P < 0.05 (relative to untreated cells).(D–I) C57BL/6 mice (three mice per group) were subjected tosubcutaneous injection of 100 µl of either vehicle(D–F) or 40 ng/ml TNF-α (G-I) and sacrificed after16 h. Skin fragments from the injection sites were fixed and costainedfor PECAM-1 (red) and L1 (green) before confocal analysis. Insets show ablood vessel cross section with the ECs positive for both PECAM-1 andL1. The arrow in F indicates an L1-positive nerve that served as aninternal control. Bars, 40 µm.
Mentions: Given the difference between human and murine immune systems, we asked whether L1is also involved in the transendothelial migration of human DCs. To this goal,we used human monocyte-derived DCs (moDCs), which express moderate levels of L1(Fig. 5 A; reference 5). The maturation of human moDCs was notaccompanied by changes in L1 levels (not depicted), confirming our observationson mouse bone marrow–derived DCs (Fig. S3 B). To evaluate the role ofL1 in transendothelial migration, CFSE-labeled moDCs were pretreated with CE7, amonoclonal antibody that has been previously shown to neutralize L1 function(21), and then allowed to cross amonolayer of TNF-α–activated human umbilical vein ECs(HUVECs). The inactivation of L1 in moDCs with CE7 resulted in a dramaticreduction of the transendothelial migration as compared with moDCs treated withan irrelevant antibody (Fig. 5 B). Giventhe expression of L1 in activated ECs (10) as well as in TNF-α–treated HUVECs (see Fig. 7 A), we also assessed thecontribution of vascular L1 to DC transendothelial migration by pretreatingHUVECs with CE7 before transmigration assays. The inactivation of endothelial L1caused a reduction in the transmigratory activity of moDCs (Fig. 5 B). Finally, when L1 was neutralized in both DCsand HUVECs, no additive effect was observed as compared with the inactivation inthe individual cell types (Fig. 5 B).Notably, CE7 had no effect on chemokine-induced migration of moDCs (notdepicted), which is in line with the results on L1-deficient mouse DCs (Fig. S4B). Thus, L1 function is required for the trafficking of human DCs through anendothelial barrier.

Bottom Line: In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo.Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration.Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs.

View Article: PubMed Central - PubMed

Affiliation: The FIRC Institute of Molecular Oncology, 20139 Milan, Italy.

ABSTRACT
The adhesion molecule L1, which is extensively characterized in the nervous system, is also expressed in dendritic cells (DCs), but its function there has remained elusive. To address this issue, we ablated L1 expression in DCs of conditional knockout mice. L1-deficient DCs were impaired in adhesion to and transmigration through monolayers of either lymphatic or blood vessel endothelial cells, implicating L1 in transendothelial migration of DCs. In agreement with these findings, L1 was expressed in cutaneous DCs that migrated to draining lymph nodes, and its ablation reduced DC trafficking in vivo. Within the skin, L1 was found in Langerhans cells but not in dermal DCs, and L1 deficiency impaired Langerhans cell migration. Under inflammatory conditions, L1 also became expressed in vascular endothelium and enhanced transmigration of DCs, likely through L1 homophilic interactions. Our results implicate L1 in the regulation of DC trafficking and shed light on novel mechanisms underlying transendothelial migration of DCs. These observations might offer novel therapeutic perspectives for the treatment of certain immunological disorders.

Show MeSH
Related in: MedlinePlus