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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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Cell uropods emerge away from the flattened cell bodies of adhered lymphocytes and are projected to the outer milieu. (a)  Confocal fluorescence microscopy analysis of the spatial orientation of the uropod and ICAM-3 localization on T lymphoblasts adhering  to ICAM-1–coated surfaces. T cells labeled with the fluorescence cytoplasmic probe CFDA-SE (green fluorescence) were allowed to  bind to coverslips coated with 10 μg/ml of ICAM-1–Fc for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19  mAb (5 μg/ml). Cells were then fixed and stained for ICAM-3 (red fluorescence) as described in Materials and Methods. Slides were analyzed by confocal laser scanning microscopy, and optical sectioning was adjusted to the plane of adhesion (A and B), 7 (C and D), and  10 μm above (E and F). Optical sections correspond to 0.3 μm in thickness. (b) T lymphoblasts were allowed to adhere to ICAM-1 in  the presence (right column) or in the absence (left column) of 10 ng/ml RANTES, and samples were processed as in a. Cytoplasmic  (green fluorescence) and ICAM-3 membrane staining (red fluorescence) are shown. Optical sectioning was adjusted to the plane of adhesion (A), 5 (B), and 8 μm above that plane (C).
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Figure 1: Cell uropods emerge away from the flattened cell bodies of adhered lymphocytes and are projected to the outer milieu. (a) Confocal fluorescence microscopy analysis of the spatial orientation of the uropod and ICAM-3 localization on T lymphoblasts adhering to ICAM-1–coated surfaces. T cells labeled with the fluorescence cytoplasmic probe CFDA-SE (green fluorescence) were allowed to bind to coverslips coated with 10 μg/ml of ICAM-1–Fc for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19 mAb (5 μg/ml). Cells were then fixed and stained for ICAM-3 (red fluorescence) as described in Materials and Methods. Slides were analyzed by confocal laser scanning microscopy, and optical sectioning was adjusted to the plane of adhesion (A and B), 7 (C and D), and 10 μm above (E and F). Optical sections correspond to 0.3 μm in thickness. (b) T lymphoblasts were allowed to adhere to ICAM-1 in the presence (right column) or in the absence (left column) of 10 ng/ml RANTES, and samples were processed as in a. Cytoplasmic (green fluorescence) and ICAM-3 membrane staining (red fluorescence) are shown. Optical sectioning was adjusted to the plane of adhesion (A), 5 (B), and 8 μm above that plane (C).

Mentions: To assess the spatial disposition of the cellular uropod and the membrane distribution of ICAM-3, confocal microscopy analysis of fluorescent-labeled (green fluorescence) T lymphoblasts, adhering to immobilized ICAM-1 was carried out in the presence of the uropod-inducing anti– ICAM-3 HP2/19 mAb. This mAb was employed since it is able to mimic the effect caused by chemokines on uropod formation (Campanero et al., 1994). We found that no ICAM-3 signal (red fluorescence) was detected in the area of contact between cells and protein substrate (Fig. 1 a, A and B). Interestingly, ICAM-3 fluorescence gradually increased on upper planes of uropods, displaying the highest intensity at a distance of 7–10 μm from the plane of the coverslip, coincident with the tips of uropods (Fig. 1 a, C–F). Similar results were obtained in experiments in which T cells were adhered to EC instead of ICAM-1 (data not shown). Moreover, the same morphological changes and ICAM-3 surface redistribution effect were observed by using chemokines. Unstimulated lymphoblasts were also stained as controls (Fig. 1 b). Hence, these results show that uropods extend away from the large flattened area of cell contact with the substratum, being well exposed to the outer milieu.


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)

Cell uropods emerge away from the flattened cell bodies of adhered lymphocytes and are projected to the outer milieu. (a)  Confocal fluorescence microscopy analysis of the spatial orientation of the uropod and ICAM-3 localization on T lymphoblasts adhering  to ICAM-1–coated surfaces. T cells labeled with the fluorescence cytoplasmic probe CFDA-SE (green fluorescence) were allowed to  bind to coverslips coated with 10 μg/ml of ICAM-1–Fc for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19  mAb (5 μg/ml). Cells were then fixed and stained for ICAM-3 (red fluorescence) as described in Materials and Methods. Slides were analyzed by confocal laser scanning microscopy, and optical sectioning was adjusted to the plane of adhesion (A and B), 7 (C and D), and  10 μm above (E and F). Optical sections correspond to 0.3 μm in thickness. (b) T lymphoblasts were allowed to adhere to ICAM-1 in  the presence (right column) or in the absence (left column) of 10 ng/ml RANTES, and samples were processed as in a. Cytoplasmic  (green fluorescence) and ICAM-3 membrane staining (red fluorescence) are shown. Optical sectioning was adjusted to the plane of adhesion (A), 5 (B), and 8 μm above that plane (C).
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Figure 1: Cell uropods emerge away from the flattened cell bodies of adhered lymphocytes and are projected to the outer milieu. (a) Confocal fluorescence microscopy analysis of the spatial orientation of the uropod and ICAM-3 localization on T lymphoblasts adhering to ICAM-1–coated surfaces. T cells labeled with the fluorescence cytoplasmic probe CFDA-SE (green fluorescence) were allowed to bind to coverslips coated with 10 μg/ml of ICAM-1–Fc for 30 min at 37°C in the presence of the uropod inducing anti–ICAM-3 HP2/19 mAb (5 μg/ml). Cells were then fixed and stained for ICAM-3 (red fluorescence) as described in Materials and Methods. Slides were analyzed by confocal laser scanning microscopy, and optical sectioning was adjusted to the plane of adhesion (A and B), 7 (C and D), and 10 μm above (E and F). Optical sections correspond to 0.3 μm in thickness. (b) T lymphoblasts were allowed to adhere to ICAM-1 in the presence (right column) or in the absence (left column) of 10 ng/ml RANTES, and samples were processed as in a. Cytoplasmic (green fluorescence) and ICAM-3 membrane staining (red fluorescence) are shown. Optical sectioning was adjusted to the plane of adhesion (A), 5 (B), and 8 μm above that plane (C).
Mentions: To assess the spatial disposition of the cellular uropod and the membrane distribution of ICAM-3, confocal microscopy analysis of fluorescent-labeled (green fluorescence) T lymphoblasts, adhering to immobilized ICAM-1 was carried out in the presence of the uropod-inducing anti– ICAM-3 HP2/19 mAb. This mAb was employed since it is able to mimic the effect caused by chemokines on uropod formation (Campanero et al., 1994). We found that no ICAM-3 signal (red fluorescence) was detected in the area of contact between cells and protein substrate (Fig. 1 a, A and B). Interestingly, ICAM-3 fluorescence gradually increased on upper planes of uropods, displaying the highest intensity at a distance of 7–10 μm from the plane of the coverslip, coincident with the tips of uropods (Fig. 1 a, C–F). Similar results were obtained in experiments in which T cells were adhered to EC instead of ICAM-1 (data not shown). Moreover, the same morphological changes and ICAM-3 surface redistribution effect were observed by using chemokines. Unstimulated lymphoblasts were also stained as controls (Fig. 1 b). Hence, these results show that uropods extend away from the large flattened area of cell contact with the substratum, being well exposed to the outer milieu.

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