Limits...
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.

Show MeSH

Related in: MedlinePlus

Involvement of  ICAM-1 and -3 in lymphocyte recruitment mediated by  cell uropods. (A) T lymphoblasts adhered for 30 min at  37°C to VCAM-1–Fc coated  surfaces in the presence of 10  ng/ml RANTES were then  incubated for 15 min with the  blocking mAb anti–ICAM-3  140.11, anti–ICAM-1 Hu5/3,  anti-CD43 TP1/36, antiCD44 HP2/9, or anti-CD45  D3/9 before the addition of a  second cohort of T cells.  Where indicated, the latter  cells were pretreated for 15  min with the anti-β2 integrin  Lia3/2 mAb, the anti-CD11a  YTH81.5 mAb, or the anti– L-selectin LAM1-3 mAb before addition to the first layer  of T cells. The recruitment index was calculated. Arithmetic mean ± 1 SD of three independent experiments is shown. (B) Cells were allowed to adhere for 30 min at 37°C to ICAM-1–Fc  coated petri dishes in the presence of the anti–ICAM-3 uropod-inducing mAb HP2/19 and additionally incubated for 15 min at 37°C either with the blocking anti–ICAM-3 mAbs 140.11 or ICR2.1 or with the anti-CD45 D3/9 mAb before addition of the second cohort of T  cells. Arithmetic mean ± 1 SD of two independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139764&req=5

Figure 7: Involvement of ICAM-1 and -3 in lymphocyte recruitment mediated by cell uropods. (A) T lymphoblasts adhered for 30 min at 37°C to VCAM-1–Fc coated surfaces in the presence of 10 ng/ml RANTES were then incubated for 15 min with the blocking mAb anti–ICAM-3 140.11, anti–ICAM-1 Hu5/3, anti-CD43 TP1/36, antiCD44 HP2/9, or anti-CD45 D3/9 before the addition of a second cohort of T cells. Where indicated, the latter cells were pretreated for 15 min with the anti-β2 integrin Lia3/2 mAb, the anti-CD11a YTH81.5 mAb, or the anti– L-selectin LAM1-3 mAb before addition to the first layer of T cells. The recruitment index was calculated. Arithmetic mean ± 1 SD of three independent experiments is shown. (B) Cells were allowed to adhere for 30 min at 37°C to ICAM-1–Fc coated petri dishes in the presence of the anti–ICAM-3 uropod-inducing mAb HP2/19 and additionally incubated for 15 min at 37°C either with the blocking anti–ICAM-3 mAbs 140.11 or ICR2.1 or with the anti-CD45 D3/9 mAb before addition of the second cohort of T cells. Arithmetic mean ± 1 SD of two independent experiments is shown.

Mentions: A panel of blocking mAbs was used to dissect the adhesion molecules involved in the lymphocyte recruitment mediated through uropods. We found that mAb against ICAM-1 and -3, two molecules concentrated in the uropod, efficiently inhibited lymphocyte recruitment (Fig. 7 A). In addition, the blockade of LFA-1, the counterreceptor for ICAM-1 and -3 (Campanero et al., 1993), also prevented these cell– cell interactions. Moreover, the YTH81.5 antibody that reacts with the I domain of the LFA-1α chain subunit and that selectively blocks the interaction with ICAM-3 but not with ICAM-1 (Landis et al., 1994) only produced a partial inhibition in cell recruitment. Interestingly, antibodies to CD43 and CD44, which are also concentrated in the uropod, as well as to CD45 that it is not redistributed (del Pozo et al., 1995) did not affect lymphocyte recruitment. Although T lymphoblasts express L-selectin (90% positive cells; mean fluorescence intensity, 188, versus negative control 5%; mean fluorescence intensity, 15), a blocking mAb directed to L-selectin did not exert any effect on cell recruitment as measured in the time-lapse videomicroscopy assay (Fig. 7 A). On the other hand, cell recruitment elicited by the HP2/19 anti–ICAM-3 mAb was abolished by coincubation with either of two different blocking anti–ICAM-3 mAbs but not with an anti-CD45 mAb (Fig. 7 B). Together, these data demonstrate that the interaction of LFA-1 integrin with its ligands ICAM-1 and -3, located in the uropod, is critical for the process of lymphocyte recruitment mediated through chemokine-induced uropods.


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)

Involvement of  ICAM-1 and -3 in lymphocyte recruitment mediated by  cell uropods. (A) T lymphoblasts adhered for 30 min at  37°C to VCAM-1–Fc coated  surfaces in the presence of 10  ng/ml RANTES were then  incubated for 15 min with the  blocking mAb anti–ICAM-3  140.11, anti–ICAM-1 Hu5/3,  anti-CD43 TP1/36, antiCD44 HP2/9, or anti-CD45  D3/9 before the addition of a  second cohort of T cells.  Where indicated, the latter  cells were pretreated for 15  min with the anti-β2 integrin  Lia3/2 mAb, the anti-CD11a  YTH81.5 mAb, or the anti– L-selectin LAM1-3 mAb before addition to the first layer  of T cells. The recruitment index was calculated. Arithmetic mean ± 1 SD of three independent experiments is shown. (B) Cells were allowed to adhere for 30 min at 37°C to ICAM-1–Fc  coated petri dishes in the presence of the anti–ICAM-3 uropod-inducing mAb HP2/19 and additionally incubated for 15 min at 37°C either with the blocking anti–ICAM-3 mAbs 140.11 or ICR2.1 or with the anti-CD45 D3/9 mAb before addition of the second cohort of T  cells. Arithmetic mean ± 1 SD of two independent experiments is shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Involvement of ICAM-1 and -3 in lymphocyte recruitment mediated by cell uropods. (A) T lymphoblasts adhered for 30 min at 37°C to VCAM-1–Fc coated surfaces in the presence of 10 ng/ml RANTES were then incubated for 15 min with the blocking mAb anti–ICAM-3 140.11, anti–ICAM-1 Hu5/3, anti-CD43 TP1/36, antiCD44 HP2/9, or anti-CD45 D3/9 before the addition of a second cohort of T cells. Where indicated, the latter cells were pretreated for 15 min with the anti-β2 integrin Lia3/2 mAb, the anti-CD11a YTH81.5 mAb, or the anti– L-selectin LAM1-3 mAb before addition to the first layer of T cells. The recruitment index was calculated. Arithmetic mean ± 1 SD of three independent experiments is shown. (B) Cells were allowed to adhere for 30 min at 37°C to ICAM-1–Fc coated petri dishes in the presence of the anti–ICAM-3 uropod-inducing mAb HP2/19 and additionally incubated for 15 min at 37°C either with the blocking anti–ICAM-3 mAbs 140.11 or ICR2.1 or with the anti-CD45 D3/9 mAb before addition of the second cohort of T cells. Arithmetic mean ± 1 SD of two independent experiments is shown.
Mentions: A panel of blocking mAbs was used to dissect the adhesion molecules involved in the lymphocyte recruitment mediated through uropods. We found that mAb against ICAM-1 and -3, two molecules concentrated in the uropod, efficiently inhibited lymphocyte recruitment (Fig. 7 A). In addition, the blockade of LFA-1, the counterreceptor for ICAM-1 and -3 (Campanero et al., 1993), also prevented these cell– cell interactions. Moreover, the YTH81.5 antibody that reacts with the I domain of the LFA-1α chain subunit and that selectively blocks the interaction with ICAM-3 but not with ICAM-1 (Landis et al., 1994) only produced a partial inhibition in cell recruitment. Interestingly, antibodies to CD43 and CD44, which are also concentrated in the uropod, as well as to CD45 that it is not redistributed (del Pozo et al., 1995) did not affect lymphocyte recruitment. Although T lymphoblasts express L-selectin (90% positive cells; mean fluorescence intensity, 188, versus negative control 5%; mean fluorescence intensity, 15), a blocking mAb directed to L-selectin did not exert any effect on cell recruitment as measured in the time-lapse videomicroscopy assay (Fig. 7 A). On the other hand, cell recruitment elicited by the HP2/19 anti–ICAM-3 mAb was abolished by coincubation with either of two different blocking anti–ICAM-3 mAbs but not with an anti-CD45 mAb (Fig. 7 B). Together, these data demonstrate that the interaction of LFA-1 integrin with its ligands ICAM-1 and -3, located in the uropod, is critical for the process of lymphocyte recruitment mediated through chemokine-induced uropods.

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