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ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes.

del Pozo MA, Cabañas C, Montoya MC, Ager A, Sánchez-Mateos P, Sánchez-Madrid F - J. Cell Biol. (1997)

Bottom Line: Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Immunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.

ABSTRACT
The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

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Effect of uropod induction on transendothelial migration of T lymphocytes. (A and B) Uropod induction increases transendothelial migration.  Migration of T lymphoblasts across confluent monolayers from a human dermal microvascular endothelial cell line (HMEC-1) was assayed in a Transwell  cell culture chamber. T lymphoblasts were allowed to bind to confluent EC  monolayers cultured on polycarbonate membranes and treated either with the  uropod-inducing mAb HP2/19 or with the noninducing anti–ICAM-3 mAb  TP1/24. A second cohort of 106 51Cr-labeled T cells was then added to the upper compartment of the chamber and incubated for long (A, from 45 min to 6 h)  and short (B, from 5 to 60 min) periods of time at 37°C in a 5% CO2 atmosphere. The percentage of cells that migrated to the lower well of the chamber  was calculated using a γ counter. A representative experiment out of five independent ones run in quadruplicate is shown. Error bars represent ± 1 SD of  values from quadruplicate Transwell chambers. (C) The increment in migration was dependent on the number of cells in the first layer of lymphocytes. In  similar experiments of transendothelial migration, different numbers of T lymphoblasts were incubated with the confluent EC monolayers, and migration of  a second cohort of 51Cr-labeled T cells was analyzed. Transmigration is presented as the ratio between percentage of migrated cells under uropod conditions and percentage of migrated cells under nonuropod conditions. Arithmethic mean ± 1 SD of three independent  experiments is shown.
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Figure 8: Effect of uropod induction on transendothelial migration of T lymphocytes. (A and B) Uropod induction increases transendothelial migration. Migration of T lymphoblasts across confluent monolayers from a human dermal microvascular endothelial cell line (HMEC-1) was assayed in a Transwell cell culture chamber. T lymphoblasts were allowed to bind to confluent EC monolayers cultured on polycarbonate membranes and treated either with the uropod-inducing mAb HP2/19 or with the noninducing anti–ICAM-3 mAb TP1/24. A second cohort of 106 51Cr-labeled T cells was then added to the upper compartment of the chamber and incubated for long (A, from 45 min to 6 h) and short (B, from 5 to 60 min) periods of time at 37°C in a 5% CO2 atmosphere. The percentage of cells that migrated to the lower well of the chamber was calculated using a γ counter. A representative experiment out of five independent ones run in quadruplicate is shown. Error bars represent ± 1 SD of values from quadruplicate Transwell chambers. (C) The increment in migration was dependent on the number of cells in the first layer of lymphocytes. In similar experiments of transendothelial migration, different numbers of T lymphoblasts were incubated with the confluent EC monolayers, and migration of a second cohort of 51Cr-labeled T cells was analyzed. Transmigration is presented as the ratio between percentage of migrated cells under uropod conditions and percentage of migrated cells under nonuropod conditions. Arithmethic mean ± 1 SD of three independent experiments is shown.

Mentions: To explore whether the enhancement in lymphocyte interactions mediated by uropods could result in an increase of the transendothelial migration of the recruited cells, we carried out migration assays using a modified Boyden chamber (Transwell®; Costar Corp.) and confluent monolayers of dermal microvascular endothelial cells (HMEC-1). We found that the induction of uropod formation in the first layer of T cells, adhered to EC, resulted in a significant enhancement of transmigration of 51Cr-labeled T cells of the second cohort (Fig. 8 A); this effect was maximal at very short time periods (45–60 min) and was detectable as early as after 15 min (Fig. 8 B). Since lymphocyte migration was similar at 4–6 h, irrespective of the addition of uropod-inducing mAb, it seems that the presence of uropods accelerates transendothelial migration rather than causes an enhancement of its overall magnitude. As expected, the enhancement of transmigration was dependent on the number of cells in the first layer of lymphocytes. Thus, 1.8 × 104 cells/ mm2 in the first layer under conditions of uropod induction, produced about a threefold enhancement in migration, compared to 4.5 × 103 lymphocytes/mm2 (Fig. 8 C).


ICAMs redistributed by chemokines to cellular uropods as a mechanism for recruitment of T lymphocytes.

del Pozo MA, Cabañas C, Montoya MC, Ager A, Sánchez-Mateos P, Sánchez-Madrid F - J. Cell Biol. (1997)

Effect of uropod induction on transendothelial migration of T lymphocytes. (A and B) Uropod induction increases transendothelial migration.  Migration of T lymphoblasts across confluent monolayers from a human dermal microvascular endothelial cell line (HMEC-1) was assayed in a Transwell  cell culture chamber. T lymphoblasts were allowed to bind to confluent EC  monolayers cultured on polycarbonate membranes and treated either with the  uropod-inducing mAb HP2/19 or with the noninducing anti–ICAM-3 mAb  TP1/24. A second cohort of 106 51Cr-labeled T cells was then added to the upper compartment of the chamber and incubated for long (A, from 45 min to 6 h)  and short (B, from 5 to 60 min) periods of time at 37°C in a 5% CO2 atmosphere. The percentage of cells that migrated to the lower well of the chamber  was calculated using a γ counter. A representative experiment out of five independent ones run in quadruplicate is shown. Error bars represent ± 1 SD of  values from quadruplicate Transwell chambers. (C) The increment in migration was dependent on the number of cells in the first layer of lymphocytes. In  similar experiments of transendothelial migration, different numbers of T lymphoblasts were incubated with the confluent EC monolayers, and migration of  a second cohort of 51Cr-labeled T cells was analyzed. Transmigration is presented as the ratio between percentage of migrated cells under uropod conditions and percentage of migrated cells under nonuropod conditions. Arithmethic mean ± 1 SD of three independent  experiments is shown.
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Figure 8: Effect of uropod induction on transendothelial migration of T lymphocytes. (A and B) Uropod induction increases transendothelial migration. Migration of T lymphoblasts across confluent monolayers from a human dermal microvascular endothelial cell line (HMEC-1) was assayed in a Transwell cell culture chamber. T lymphoblasts were allowed to bind to confluent EC monolayers cultured on polycarbonate membranes and treated either with the uropod-inducing mAb HP2/19 or with the noninducing anti–ICAM-3 mAb TP1/24. A second cohort of 106 51Cr-labeled T cells was then added to the upper compartment of the chamber and incubated for long (A, from 45 min to 6 h) and short (B, from 5 to 60 min) periods of time at 37°C in a 5% CO2 atmosphere. The percentage of cells that migrated to the lower well of the chamber was calculated using a γ counter. A representative experiment out of five independent ones run in quadruplicate is shown. Error bars represent ± 1 SD of values from quadruplicate Transwell chambers. (C) The increment in migration was dependent on the number of cells in the first layer of lymphocytes. In similar experiments of transendothelial migration, different numbers of T lymphoblasts were incubated with the confluent EC monolayers, and migration of a second cohort of 51Cr-labeled T cells was analyzed. Transmigration is presented as the ratio between percentage of migrated cells under uropod conditions and percentage of migrated cells under nonuropod conditions. Arithmethic mean ± 1 SD of three independent experiments is shown.
Mentions: To explore whether the enhancement in lymphocyte interactions mediated by uropods could result in an increase of the transendothelial migration of the recruited cells, we carried out migration assays using a modified Boyden chamber (Transwell®; Costar Corp.) and confluent monolayers of dermal microvascular endothelial cells (HMEC-1). We found that the induction of uropod formation in the first layer of T cells, adhered to EC, resulted in a significant enhancement of transmigration of 51Cr-labeled T cells of the second cohort (Fig. 8 A); this effect was maximal at very short time periods (45–60 min) and was detectable as early as after 15 min (Fig. 8 B). Since lymphocyte migration was similar at 4–6 h, irrespective of the addition of uropod-inducing mAb, it seems that the presence of uropods accelerates transendothelial migration rather than causes an enhancement of its overall magnitude. As expected, the enhancement of transmigration was dependent on the number of cells in the first layer of lymphocytes. Thus, 1.8 × 104 cells/ mm2 in the first layer under conditions of uropod induction, produced about a threefold enhancement in migration, compared to 4.5 × 103 lymphocytes/mm2 (Fig. 8 C).

Bottom Line: Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment.Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process.An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation.

View Article: PubMed Central - PubMed

Affiliation: Servicio de Immunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.

ABSTRACT
The recruitment of leukocytes from the bloodstream is a key step in the inflammatory reaction, and chemokines are among the main regulators of this process. During lymphocyte-endothelial interaction, chemokines induce the polarization of T lymphocytes, with the formation of a cytoplasmic projection (uropod) and redistribution of several adhesion molecules (ICAM-1,-3, CD43, CD44) to this structure. Although it has been reported that these cytokines regulate the adhesive state of integrins in leukocytes, their precise mechanisms of chemoattraction remain to be elucidated. We have herein studied the functional role of the lymphocyte uropod. Confocal microscopy studies clearly showed that cell uropods project away from the cell bodies of adhered lymphocytes and that polarized T cells contact other T cells through the uropod structure. Time-lapse videomicroscopy studies revealed that uropod-bearing T cells were able, through this cellular projection, to contact, capture, and transport additional bystander T cells. Quantitative analysis revealed that the induction of uropods results in a 5-10-fold increase in cell recruitment. Uropod-mediated cell recruitment seems to have physiological relevance, since it was promoted by both CD45R0+ peripheral blood memory T cells as well as by in vivo activated lymphocytes. Additional studies showed that the cell recruitment mediated by uropods was abrogated with antibodies to ICAM-1, -3, and LFA-1, whereas mAb to CD43, CD44, CD45, and L-selectin did not have a significant effect, thus indicating that the interaction of LFA-1 with ICAM-1 and -3 appears to be responsible for this process. To determine whether the increment in cell recruitment mediated by uropod may affect the transendothelial migration of T cells, we carried out chemotaxis assays through confluent monolayers of endothelial cells specialized in lymphocyte extravasation. An enhancement of T cell migration was observed under conditions of uropod formation, and this increase was prevented by incubation with either blocking anti-ICAM-3 mAbs or drugs that impair uropod formation. These data indicate that the cell interactions mediated by cell uropods represent a cooperative mechanism in lymphocyte recruitment, which may act as an amplification system in the inflammatory response.

Show MeSH
Related in: MedlinePlus