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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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T cells are recruited by uropod-bearing T lymphocytes. Time-lapse videomicroscopy analysis of lymphocyte–lymphocyte interactions. (A and B) The chemokine RANTES and the uropod-inducing anti–ICAM-3 HP2/19 mAb induce T cells to contact, capture,  and transport other T cells through their uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with ICAM-1–Fc  and treated with the anti–ICAM-3 uropod-inducing mAb HP2/19 (Ab, 5 μg/ml), with the isotype-matched nonuropod inducing anti– ICAM-3 TP1/24 (Aa, 5 μg/ml), or with 10 ng/ml RANTES (B) for 30 min at 37°C. After addition of a second cohort of untreated T lymphocytes (exhibiting a phase-bright appearance readily distinguishable from the phase-dark cells of the first cohort), cell–cell interactions were filmed with a time-lapse videocassette recorder for 1 h. Time frames obtained from videotape recording of one representative  experiment are shown. White arrowheads point to uropods displayed by phase-dark cells of the first layer; black arrows indicate phasebright T lymphocytes captured by uropod-bearing cells, while the small white arrows mark the direction of movement of the adhered  cell. (Inset) Polarized lymphocyte migrating on ICAM-1–coated surface, showing the phase-dark leading edge (L) and the phase-bright  uropod (U). (C) Measurement of cell recruitment mediated by cell uropods. T lymphoblasts were allowed to bind to plastic petri dishes  coated with VCAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP-1α, MIP-1β, IL-8, 20 ng/ ml PMA, or 5 μg/ml anti–ICAM-3 HP2/19 and TP1/24 mAb. After addition of a second cohort of T cells, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of three independent experiments is shown.
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Figure 3: T cells are recruited by uropod-bearing T lymphocytes. Time-lapse videomicroscopy analysis of lymphocyte–lymphocyte interactions. (A and B) The chemokine RANTES and the uropod-inducing anti–ICAM-3 HP2/19 mAb induce T cells to contact, capture, and transport other T cells through their uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with ICAM-1–Fc and treated with the anti–ICAM-3 uropod-inducing mAb HP2/19 (Ab, 5 μg/ml), with the isotype-matched nonuropod inducing anti– ICAM-3 TP1/24 (Aa, 5 μg/ml), or with 10 ng/ml RANTES (B) for 30 min at 37°C. After addition of a second cohort of untreated T lymphocytes (exhibiting a phase-bright appearance readily distinguishable from the phase-dark cells of the first cohort), cell–cell interactions were filmed with a time-lapse videocassette recorder for 1 h. Time frames obtained from videotape recording of one representative experiment are shown. White arrowheads point to uropods displayed by phase-dark cells of the first layer; black arrows indicate phasebright T lymphocytes captured by uropod-bearing cells, while the small white arrows mark the direction of movement of the adhered cell. (Inset) Polarized lymphocyte migrating on ICAM-1–coated surface, showing the phase-dark leading edge (L) and the phase-bright uropod (U). (C) Measurement of cell recruitment mediated by cell uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with VCAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP-1α, MIP-1β, IL-8, 20 ng/ ml PMA, or 5 μg/ml anti–ICAM-3 HP2/19 and TP1/24 mAb. After addition of a second cohort of T cells, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of three independent experiments is shown.

Mentions: Since confocal microscopy analysis only provided a static view of the possible functional role of uropod, we decided to undertake a dynamic assessment of this issue through a time-lapse phase contrast microscopy analysis. A first layer of T cells was allowed to attach to and spread on an ICAM-1–coated surface, and then these cells were induced to develop uropods with the HP2/19 anti–ICAM-3 mAb or different chemokines. A second cohort of T cells was then added, and cell–cell interactions were filmed. Attached lymphocytes in the first cohort adopted a phasedark morphology. Under conditions of uropod induction, a significant proportion of cells of the second cohort, which exhibited a phase-bright appearance readily distinguishable from the first cohort, were contacted, trapped, and finally engaged by cells of the first cohort (Fig. 3, A and B). Interestingly, the adhered lymphocytes of the first cohort were moving on the ICAM-1–coated surface, displaying the uropod in the antipodean pole relative to the advancing front of the cell, and were transporting other T cells engaged through the uropod (Fig. 3 B). By counting the number of cells of the second cohort that were recruited by cells of the first layer, it was estimated that the induction of the uropod resulted in a 5–10-fold increase in cell recruitment (Fig. 3 C). In contrast, other stimulating agents, such as phorbol esters, that promote integrin activation but are unable to trigger uropod formation, failed to stimulate T cell recruitment (Fig. 3 C), thus underlining the participation of uropods in this phenomenon.


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)

T cells are recruited by uropod-bearing T lymphocytes. Time-lapse videomicroscopy analysis of lymphocyte–lymphocyte interactions. (A and B) The chemokine RANTES and the uropod-inducing anti–ICAM-3 HP2/19 mAb induce T cells to contact, capture,  and transport other T cells through their uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with ICAM-1–Fc  and treated with the anti–ICAM-3 uropod-inducing mAb HP2/19 (Ab, 5 μg/ml), with the isotype-matched nonuropod inducing anti– ICAM-3 TP1/24 (Aa, 5 μg/ml), or with 10 ng/ml RANTES (B) for 30 min at 37°C. After addition of a second cohort of untreated T lymphocytes (exhibiting a phase-bright appearance readily distinguishable from the phase-dark cells of the first cohort), cell–cell interactions were filmed with a time-lapse videocassette recorder for 1 h. Time frames obtained from videotape recording of one representative  experiment are shown. White arrowheads point to uropods displayed by phase-dark cells of the first layer; black arrows indicate phasebright T lymphocytes captured by uropod-bearing cells, while the small white arrows mark the direction of movement of the adhered  cell. (Inset) Polarized lymphocyte migrating on ICAM-1–coated surface, showing the phase-dark leading edge (L) and the phase-bright  uropod (U). (C) Measurement of cell recruitment mediated by cell uropods. T lymphoblasts were allowed to bind to plastic petri dishes  coated with VCAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP-1α, MIP-1β, IL-8, 20 ng/ ml PMA, or 5 μg/ml anti–ICAM-3 HP2/19 and TP1/24 mAb. After addition of a second cohort of T cells, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of three independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139764&req=5

Figure 3: T cells are recruited by uropod-bearing T lymphocytes. Time-lapse videomicroscopy analysis of lymphocyte–lymphocyte interactions. (A and B) The chemokine RANTES and the uropod-inducing anti–ICAM-3 HP2/19 mAb induce T cells to contact, capture, and transport other T cells through their uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with ICAM-1–Fc and treated with the anti–ICAM-3 uropod-inducing mAb HP2/19 (Ab, 5 μg/ml), with the isotype-matched nonuropod inducing anti– ICAM-3 TP1/24 (Aa, 5 μg/ml), or with 10 ng/ml RANTES (B) for 30 min at 37°C. After addition of a second cohort of untreated T lymphocytes (exhibiting a phase-bright appearance readily distinguishable from the phase-dark cells of the first cohort), cell–cell interactions were filmed with a time-lapse videocassette recorder for 1 h. Time frames obtained from videotape recording of one representative experiment are shown. White arrowheads point to uropods displayed by phase-dark cells of the first layer; black arrows indicate phasebright T lymphocytes captured by uropod-bearing cells, while the small white arrows mark the direction of movement of the adhered cell. (Inset) Polarized lymphocyte migrating on ICAM-1–coated surface, showing the phase-dark leading edge (L) and the phase-bright uropod (U). (C) Measurement of cell recruitment mediated by cell uropods. T lymphoblasts were allowed to bind to plastic petri dishes coated with VCAM-1–Fc for 30 min at 37°C in the presence of medium alone, 10 ng/ml RANTES, MCP-1, MIP-1α, MIP-1β, IL-8, 20 ng/ ml PMA, or 5 μg/ml anti–ICAM-3 HP2/19 and TP1/24 mAb. After addition of a second cohort of T cells, cell–cell interactions were recorded for 1 h, and the recruitment index was estimated as described in Materials and Methods. Arithmetic mean ± 1 SD of three independent experiments is shown.
Mentions: Since confocal microscopy analysis only provided a static view of the possible functional role of uropod, we decided to undertake a dynamic assessment of this issue through a time-lapse phase contrast microscopy analysis. A first layer of T cells was allowed to attach to and spread on an ICAM-1–coated surface, and then these cells were induced to develop uropods with the HP2/19 anti–ICAM-3 mAb or different chemokines. A second cohort of T cells was then added, and cell–cell interactions were filmed. Attached lymphocytes in the first cohort adopted a phasedark morphology. Under conditions of uropod induction, a significant proportion of cells of the second cohort, which exhibited a phase-bright appearance readily distinguishable from the first cohort, were contacted, trapped, and finally engaged by cells of the first cohort (Fig. 3, A and B). Interestingly, the adhered lymphocytes of the first cohort were moving on the ICAM-1–coated surface, displaying the uropod in the antipodean pole relative to the advancing front of the cell, and were transporting other T cells engaged through the uropod (Fig. 3 B). By counting the number of cells of the second cohort that were recruited by cells of the first layer, it was estimated that the induction of the uropod resulted in a 5–10-fold increase in cell recruitment (Fig. 3 C). In contrast, other stimulating agents, such as phorbol esters, that promote integrin activation but are unable to trigger uropod formation, failed to stimulate T cell recruitment (Fig. 3 C), thus underlining the participation of uropods in this phenomenon.

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