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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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Calpain inhibitors block LFA-1–mediated adhesion induced by agonists acting intracellularly. Cells were preincubated  with or without 280 μM (100 μg/ml) calpeptin for 30 min at 37°C  before analysis in a T cell adhesion assay where stimulants were  50 nM PdBu, 5 μM thapsigargin, 10 μg/ml anti-CD3 mAb G19.4,  5 mM Mg2+/1 mM EGTA.
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Figure 6: Calpain inhibitors block LFA-1–mediated adhesion induced by agonists acting intracellularly. Cells were preincubated with or without 280 μM (100 μg/ml) calpeptin for 30 min at 37°C before analysis in a T cell adhesion assay where stimulants were 50 nM PdBu, 5 μM thapsigargin, 10 μg/ml anti-CD3 mAb G19.4, 5 mM Mg2+/1 mM EGTA.

Mentions: Another enzyme activated by Ca2+ is calpain, a multifunctional protease that is located in the cytosol (Sorimachi et al., 1994). We monitored LFA-1 adhesion stimulated by thapsigargin, PdBu, XL-TCR–CD3, and Mg2+/EGTA after preincubation with the membrane permeable calpain inhibitor, calpeptin (Tsujinaka et al., 1988; Kwak et al., 1993). This agent caused maximal inhibition of T cell adhesion at 100 μg/ml (280 μM) after stimulation by thapsigargin, PdBu, and XL-TCR–CD3, but had no effect on Mg2+-induced adhesion (Fig. 6). A further calpain inhibitor, CBZ-LVG, also blocked adhesion at similar concentrations (data not shown). There was no alteration in cell viability at concentrations at which these inhibitors were maximally active. We next found that the levels of clustered LFA-1 detected after treatment with thapsigargin (Fig. 7 a), XL-CD3 (Fig. 7 b), and PdBu (data not shown) were diminished by calpeptin treatment (i.e., there is less fluorescence highlighted [red] in Fig. 7 c than in a and in Fig. 7 d than in b) to levels similar to those on resting T cells (Table I, Free membrane).


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)

Calpain inhibitors block LFA-1–mediated adhesion induced by agonists acting intracellularly. Cells were preincubated  with or without 280 μM (100 μg/ml) calpeptin for 30 min at 37°C  before analysis in a T cell adhesion assay where stimulants were  50 nM PdBu, 5 μM thapsigargin, 10 μg/ml anti-CD3 mAb G19.4,  5 mM Mg2+/1 mM EGTA.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Calpain inhibitors block LFA-1–mediated adhesion induced by agonists acting intracellularly. Cells were preincubated with or without 280 μM (100 μg/ml) calpeptin for 30 min at 37°C before analysis in a T cell adhesion assay where stimulants were 50 nM PdBu, 5 μM thapsigargin, 10 μg/ml anti-CD3 mAb G19.4, 5 mM Mg2+/1 mM EGTA.
Mentions: Another enzyme activated by Ca2+ is calpain, a multifunctional protease that is located in the cytosol (Sorimachi et al., 1994). We monitored LFA-1 adhesion stimulated by thapsigargin, PdBu, XL-TCR–CD3, and Mg2+/EGTA after preincubation with the membrane permeable calpain inhibitor, calpeptin (Tsujinaka et al., 1988; Kwak et al., 1993). This agent caused maximal inhibition of T cell adhesion at 100 μg/ml (280 μM) after stimulation by thapsigargin, PdBu, and XL-TCR–CD3, but had no effect on Mg2+-induced adhesion (Fig. 6). A further calpain inhibitor, CBZ-LVG, also blocked adhesion at similar concentrations (data not shown). There was no alteration in cell viability at concentrations at which these inhibitors were maximally active. We next found that the levels of clustered LFA-1 detected after treatment with thapsigargin (Fig. 7 a), XL-CD3 (Fig. 7 b), and PdBu (data not shown) were diminished by calpeptin treatment (i.e., there is less fluorescence highlighted [red] in Fig. 7 c than in a and in Fig. 7 d than in b) to levels similar to those on resting T cells (Table I, Free membrane).

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