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LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain.

Stewart MP, McDowall A, Hogg N - J. Cell Biol. (1998)

Bottom Line: The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues.We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane.We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity.

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

ABSTRACT
The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2, 5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)-CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.

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Thapsigargin stimulates LFA-1 clustering through a cytoskeletal release mechanism. T cells adhered onto poly-L-lysine  were either (a) treated with 5 μM thapsigargin for 30 min at 37°C,  or (b) preincubated with 1 μM jasplakinolide for 30 min at 37°C,  before treatment as in a. LFA-1 was detected with FITC-conjugated LFA-1 specific mAb and analyzed by confocal microscopy  as in Fig. 4. Bar, 10 μm.
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Figure 5: Thapsigargin stimulates LFA-1 clustering through a cytoskeletal release mechanism. T cells adhered onto poly-L-lysine were either (a) treated with 5 μM thapsigargin for 30 min at 37°C, or (b) preincubated with 1 μM jasplakinolide for 30 min at 37°C, before treatment as in a. LFA-1 was detected with FITC-conjugated LFA-1 specific mAb and analyzed by confocal microscopy as in Fig. 4. Bar, 10 μm.

Mentions: We next used confocal microscopy to ask whether jasplakinolide affected the distribution of LFA-1 after pretreatment of thapsigargin-stimulated T cells. As seen in Fig. 5, jasplakinolide diminished the fluorescence levels of LFA-1 detected by confocal microscopy (i.e., there is less fluorescence highlighted [red] in Fig. 5 b than in a). Identical levels of LFA-1 expression were detected by flow cytometry before and after jasplakinolide treatment (data not shown), indicating that the inhibitor interfered with LFA-1 clustering rather than causing receptor loss. Similar results were obtained after T cell stimulation with phorbol ester and through CD3–TCR (data not shown). Therefore, clustering of LFA-1 on the membrane is dependent on the disassembly of the actin fibers, suggesting that adhesion through LFA-1 clustering is promoted by active release from restraints imposed by the cytoskeleton.


LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain.

Stewart MP, McDowall A, Hogg N - J. Cell Biol. (1998)

Thapsigargin stimulates LFA-1 clustering through a cytoskeletal release mechanism. T cells adhered onto poly-L-lysine  were either (a) treated with 5 μM thapsigargin for 30 min at 37°C,  or (b) preincubated with 1 μM jasplakinolide for 30 min at 37°C,  before treatment as in a. LFA-1 was detected with FITC-conjugated LFA-1 specific mAb and analyzed by confocal microscopy  as in Fig. 4. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140165&req=5

Figure 5: Thapsigargin stimulates LFA-1 clustering through a cytoskeletal release mechanism. T cells adhered onto poly-L-lysine were either (a) treated with 5 μM thapsigargin for 30 min at 37°C, or (b) preincubated with 1 μM jasplakinolide for 30 min at 37°C, before treatment as in a. LFA-1 was detected with FITC-conjugated LFA-1 specific mAb and analyzed by confocal microscopy as in Fig. 4. Bar, 10 μm.
Mentions: We next used confocal microscopy to ask whether jasplakinolide affected the distribution of LFA-1 after pretreatment of thapsigargin-stimulated T cells. As seen in Fig. 5, jasplakinolide diminished the fluorescence levels of LFA-1 detected by confocal microscopy (i.e., there is less fluorescence highlighted [red] in Fig. 5 b than in a). Identical levels of LFA-1 expression were detected by flow cytometry before and after jasplakinolide treatment (data not shown), indicating that the inhibitor interfered with LFA-1 clustering rather than causing receptor loss. Similar results were obtained after T cell stimulation with phorbol ester and through CD3–TCR (data not shown). Therefore, clustering of LFA-1 on the membrane is dependent on the disassembly of the actin fibers, suggesting that adhesion through LFA-1 clustering is promoted by active release from restraints imposed by the cytoskeleton.

Bottom Line: The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues.We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane.We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity.

View Article: PubMed Central - PubMed

Affiliation: Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.

ABSTRACT
The activity of integrins on leukocytes is kept under tight control to avoid inappropriate adhesion while these cells are circulating in blood or migrating through tissues. Using lymphocyte function-associated antigen-1 (LFA-1) on T cells as a model, we have investigated adhesion to ligand intercellular adhesion molecule-1 induced by the Ca2+ mobilizers, ionomycin, 2, 5-di-t-butylhydroquinone, and thapsigargin, and the well studied stimulators such as phorbol ester and cross-linking of the antigen-specific T cell receptor (TCR)-CD3 complex. We report here that after exposure of T cells to these agonists, integrin is released from cytoskeletal control by the Ca2+-induced activation of a calpain-like enzyme, and adhesive contact between cells is strengthened by means of the clustering of mobilized LFA-1 on the membrane. We propose that methods of leukocyte stimulation that cause Ca2+ fluxes induce LFA-1 adhesion by regulation of calpain activity. These findings suggest a mechanism whereby engagement of the TCR could promote adhesion strengthening at an early stage of interaction with an antigen-presenting cell.

Show MeSH
Related in: MedlinePlus