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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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Lymphocyte polarization occurs in vivo. (a) Lymphocyte polarization and  ICAM-3 redistribution to the  cellular uropod during TIL  binding to autologous melanoma tumor cells. TIL were  cocultured with a monolayer  of melanoma cells from the  same patient for 1 h at 37°C.  Fixed cells were then stained  for ICAM-3 with TP1/24 mAb  followed by incubation with a  Cy3-goat anti–mouse IgG, as  described in Materials and  Methods. Epifluorescent and  bright field conditions were  photographed on the same  frame by double exposure. Arrows point to uropods. (b) Tissue distribution of ICAM-3 in  T lymphocytes that infiltrate a  tumor specimen. A tissue section of a lung metastatic melanoma biopsy was double immunofluorescence stained for  ICAM-3 (red fluorescence)  and CD3 (green fluorescence)  as described in Materials and  Methods. Tissue sections were  analyzed by confocal laser  scanning microscopy. Serial  optical sections of 0.5 μm thick  are shown (from A to E). A  small aggregate of three lymphocytes connected through  the tissue is followed. Arrows  point out the area of cell–cell  contact where ICAM-3 is concentrated.
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Figure 5: Lymphocyte polarization occurs in vivo. (a) Lymphocyte polarization and ICAM-3 redistribution to the cellular uropod during TIL binding to autologous melanoma tumor cells. TIL were cocultured with a monolayer of melanoma cells from the same patient for 1 h at 37°C. Fixed cells were then stained for ICAM-3 with TP1/24 mAb followed by incubation with a Cy3-goat anti–mouse IgG, as described in Materials and Methods. Epifluorescent and bright field conditions were photographed on the same frame by double exposure. Arrows point to uropods. (b) Tissue distribution of ICAM-3 in T lymphocytes that infiltrate a tumor specimen. A tissue section of a lung metastatic melanoma biopsy was double immunofluorescence stained for ICAM-3 (red fluorescence) and CD3 (green fluorescence) as described in Materials and Methods. Tissue sections were analyzed by confocal laser scanning microscopy. Serial optical sections of 0.5 μm thick are shown (from A to E). A small aggregate of three lymphocytes connected through the tissue is followed. Arrows point out the area of cell–cell contact where ICAM-3 is concentrated.

Mentions: We also studied lymphocyte recruitment with physiologically in vivo TIL. These lymphocytes, which infiltrate different tumors, display an in vivo activated phenotype (CD45R0+ CD69+) and high levels of cytotoxicity against autologous tumor cells (Itoh et al., 1988). Freshly prepared TIL adhered to autologous melanoma tumor cells displayed a polarized morphology with ICAM-3 redistribution to the cell uropod (Fig. 5 a). Moreover, TIL were found to be polarized, and ICAM-3 was found to be concentrated in the area of cell contact in the small aggregates of T cells that infiltrate the melanoma tissue sections (Fig. 5 b). TIL were able to induce T cell recruitment when adhered to either ICAM-1– or FN80-coated surfaces (Fig. 6 a); this phenomenon was also observed when TIL adhered to a monolayer of the tumor cells that these lymphocytes infiltrated in vivo (Fig. 6 b). Additionally, we also explored the in vivo relevance of this phenomenon in the inflammatory disease rheumatoid arthritis. Lymphocytes isolated from either peripheral blood or synovial fluid of these patients were polarized when adhered to ICAM-1 (20 and 77%, respectively) and consequently, were able to recruit peripheral blood lymphocytes of the same patient (recruitment indexes: 0.13 and 0.37, respectively).


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)

Lymphocyte polarization occurs in vivo. (a) Lymphocyte polarization and  ICAM-3 redistribution to the  cellular uropod during TIL  binding to autologous melanoma tumor cells. TIL were  cocultured with a monolayer  of melanoma cells from the  same patient for 1 h at 37°C.  Fixed cells were then stained  for ICAM-3 with TP1/24 mAb  followed by incubation with a  Cy3-goat anti–mouse IgG, as  described in Materials and  Methods. Epifluorescent and  bright field conditions were  photographed on the same  frame by double exposure. Arrows point to uropods. (b) Tissue distribution of ICAM-3 in  T lymphocytes that infiltrate a  tumor specimen. A tissue section of a lung metastatic melanoma biopsy was double immunofluorescence stained for  ICAM-3 (red fluorescence)  and CD3 (green fluorescence)  as described in Materials and  Methods. Tissue sections were  analyzed by confocal laser  scanning microscopy. Serial  optical sections of 0.5 μm thick  are shown (from A to E). A  small aggregate of three lymphocytes connected through  the tissue is followed. Arrows  point out the area of cell–cell  contact where ICAM-3 is concentrated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139764&req=5

Figure 5: Lymphocyte polarization occurs in vivo. (a) Lymphocyte polarization and ICAM-3 redistribution to the cellular uropod during TIL binding to autologous melanoma tumor cells. TIL were cocultured with a monolayer of melanoma cells from the same patient for 1 h at 37°C. Fixed cells were then stained for ICAM-3 with TP1/24 mAb followed by incubation with a Cy3-goat anti–mouse IgG, as described in Materials and Methods. Epifluorescent and bright field conditions were photographed on the same frame by double exposure. Arrows point to uropods. (b) Tissue distribution of ICAM-3 in T lymphocytes that infiltrate a tumor specimen. A tissue section of a lung metastatic melanoma biopsy was double immunofluorescence stained for ICAM-3 (red fluorescence) and CD3 (green fluorescence) as described in Materials and Methods. Tissue sections were analyzed by confocal laser scanning microscopy. Serial optical sections of 0.5 μm thick are shown (from A to E). A small aggregate of three lymphocytes connected through the tissue is followed. Arrows point out the area of cell–cell contact where ICAM-3 is concentrated.
Mentions: We also studied lymphocyte recruitment with physiologically in vivo TIL. These lymphocytes, which infiltrate different tumors, display an in vivo activated phenotype (CD45R0+ CD69+) and high levels of cytotoxicity against autologous tumor cells (Itoh et al., 1988). Freshly prepared TIL adhered to autologous melanoma tumor cells displayed a polarized morphology with ICAM-3 redistribution to the cell uropod (Fig. 5 a). Moreover, TIL were found to be polarized, and ICAM-3 was found to be concentrated in the area of cell contact in the small aggregates of T cells that infiltrate the melanoma tissue sections (Fig. 5 b). TIL were able to induce T cell recruitment when adhered to either ICAM-1– or FN80-coated surfaces (Fig. 6 a); this phenomenon was also observed when TIL adhered to a monolayer of the tumor cells that these lymphocytes infiltrated in vivo (Fig. 6 b). Additionally, we also explored the in vivo relevance of this phenomenon in the inflammatory disease rheumatoid arthritis. Lymphocytes isolated from either peripheral blood or synovial fluid of these patients were polarized when adhered to ICAM-1 (20 and 77%, respectively) and consequently, were able to recruit peripheral blood lymphocytes of the same patient (recruitment indexes: 0.13 and 0.37, respectively).

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