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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, ionomcyin, and dBHQ do not induce  binding of soluble ICAM-1 to LFA-1. Soluble ICAM-1Fc at 1  mg/ml (or 4.5 μM) was incubated with thapsigargin (5 μM), ionomcyin (0.7 μM), dBHQ (50 μM), PdBu (50 nM), and Mg2+/ EGTA (5 mM/1 mM)–stimulated cells for 30 min at 37°C (Stewart et al., 1996). Bound sICAM-1 was detected with FITC-conjugated goat anti–human (Fc specific) Ab and analyzed by flow cytometry. Results are expressed as median fluorescence intensity.  An experiment representative of three is presented.
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Figure 3: Thapsigargin, ionomcyin, and dBHQ do not induce binding of soluble ICAM-1 to LFA-1. Soluble ICAM-1Fc at 1 mg/ml (or 4.5 μM) was incubated with thapsigargin (5 μM), ionomcyin (0.7 μM), dBHQ (50 μM), PdBu (50 nM), and Mg2+/ EGTA (5 mM/1 mM)–stimulated cells for 30 min at 37°C (Stewart et al., 1996). Bound sICAM-1 was detected with FITC-conjugated goat anti–human (Fc specific) Ab and analyzed by flow cytometry. Results are expressed as median fluorescence intensity. An experiment representative of three is presented.

Mentions: To investigate the mechanism of Ca2+ action, the characteristics of LFA-1–mediated adhesion caused by the Ca2+ mobilizers was examined. One question of interest was whether the Ca2+-mediated signaling increased the ability of LFA-1 to bind soluble ICAM-1 (sICAM-1). In a previous study, sICAM-1 binding distinguished high affinity Mg2+-stimulated LFA-1 from low affinity LFA-1 on phorbol ester-stimulated and XL-TCR–CD3 T cells (Stewart et al., 1996). None of the Ca2+ mobilizers showed any induction of sICAM-1 binding even at sICAM-1 levels of 1 mg/ml (4.5 μM), in contrast to the enhanced ability of Mg2+-stimulated T cells to bind sICAM-1, which served as a positive control (Fig. 3). It can be concluded that the Ca2+ mobilizers resemble the phorbol ester or TCR–CD3 model of adhesion by failing to promote an increase in the ability of LFA-1 to bind to soluble ICAM-1.


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, ionomcyin, and dBHQ do not induce  binding of soluble ICAM-1 to LFA-1. Soluble ICAM-1Fc at 1  mg/ml (or 4.5 μM) was incubated with thapsigargin (5 μM), ionomcyin (0.7 μM), dBHQ (50 μM), PdBu (50 nM), and Mg2+/ EGTA (5 mM/1 mM)–stimulated cells for 30 min at 37°C (Stewart et al., 1996). Bound sICAM-1 was detected with FITC-conjugated goat anti–human (Fc specific) Ab and analyzed by flow cytometry. Results are expressed as median fluorescence intensity.  An experiment representative of three is presented.
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Related In: Results  -  Collection

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

Figure 3: Thapsigargin, ionomcyin, and dBHQ do not induce binding of soluble ICAM-1 to LFA-1. Soluble ICAM-1Fc at 1 mg/ml (or 4.5 μM) was incubated with thapsigargin (5 μM), ionomcyin (0.7 μM), dBHQ (50 μM), PdBu (50 nM), and Mg2+/ EGTA (5 mM/1 mM)–stimulated cells for 30 min at 37°C (Stewart et al., 1996). Bound sICAM-1 was detected with FITC-conjugated goat anti–human (Fc specific) Ab and analyzed by flow cytometry. Results are expressed as median fluorescence intensity. An experiment representative of three is presented.
Mentions: To investigate the mechanism of Ca2+ action, the characteristics of LFA-1–mediated adhesion caused by the Ca2+ mobilizers was examined. One question of interest was whether the Ca2+-mediated signaling increased the ability of LFA-1 to bind soluble ICAM-1 (sICAM-1). In a previous study, sICAM-1 binding distinguished high affinity Mg2+-stimulated LFA-1 from low affinity LFA-1 on phorbol ester-stimulated and XL-TCR–CD3 T cells (Stewart et al., 1996). None of the Ca2+ mobilizers showed any induction of sICAM-1 binding even at sICAM-1 levels of 1 mg/ml (4.5 μM), in contrast to the enhanced ability of Mg2+-stimulated T cells to bind sICAM-1, which served as a positive control (Fig. 3). It can be concluded that the Ca2+ mobilizers resemble the phorbol ester or TCR–CD3 model of adhesion by failing to promote an increase in the ability of LFA-1 to bind to soluble ICAM-1.

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