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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. T cells adhered onto poly-L-lysine were (a) unstimulated; (b) treated with  5 mM Mg2+/1 mM EGTA; or (c) treated with 5 μM thapsigargin  for 30 min at 37°C. LFA-1 was detected with FITC-conjugated  LFA-1–specific mAb and analyzed by confocal microscopy. Red  color indicates fluorescence intensity exceeding a preset pixel  value (see Materials and Methods). This setting remained constant between samples. Bar, 10 μm.
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Figure 4: Thapsigargin stimulates LFA-1 clustering. T cells adhered onto poly-L-lysine were (a) unstimulated; (b) treated with 5 mM Mg2+/1 mM EGTA; or (c) treated with 5 μM thapsigargin for 30 min at 37°C. LFA-1 was detected with FITC-conjugated LFA-1–specific mAb and analyzed by confocal microscopy. Red color indicates fluorescence intensity exceeding a preset pixel value (see Materials and Methods). This setting remained constant between samples. Bar, 10 μm.

Mentions: As the Ca2+ mobilizers did not cause a detectable increase in the affinity of LFA-1, we used confocal microscopy to investigate whether the membrane distribution of LFA-1 was altered in a manner facilitating adhesion. To analyze LFA-1 distribution, we highlighted the confocal microscopy images such that the membrane regions with the highest LFA-1 fluorescence (i.e., in the pixel intensity range 150–250, where 250 is the highest possible value) are depicted in red and all others in white (Fig. 4). Unstimulated cells (Fig. 4 a) and Mg2+/EGTA–stimulated cells (Fig. 4 b) have very little high intensity LFA-1 fluorescence (red) in comparison to that of thapsigargin-stimulated cells (Fig. 4 c). These observations were confirmed by quantifying (see Materials and Methods) the levels of fluorescence for each sample on regions of the membrane where there was no contact between cells (Table I, Free membrane). Statistical analysis (one way ANOVA) of these measurements confirmed that the level of fluorescence on the thapsigargin-stimulated cells, but not on the Mg2+/EGTA–stimulated T cells, was significantly increased compared to the level on the resting T cells (Table I, Significance levels). This increase in the intensity of LFA-1 fluorescence upon thapsigargin stimulation was detected with three distinct CD11a mAbs, 38, F110.22, and G25.2 (data not shown), and therefore does not represent the exposure of a particular epitope. These results suggested that thapsigargin might stimulate an increase in the cell surface expression of LFA-1. When measured by flow cytometry, however, results revealed that the level of LFA-1 expression after thapsigargin stimulation did not differ from LFA-1 expression on the resting or Mg2+/EGTA–treated cells (Table I, Flow cytometry). This suggested that the increase in signal strength observed by confocal microscopy upon thapsigargin stimulation reflects increased clustering that creates a higher LFA-1 fluorescence intensity. Similar results were obtained with the other Ca2+-mobilizing agents, with PdBu, and by XL-TCR–CD3 (data not shown).


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. T cells adhered onto poly-L-lysine were (a) unstimulated; (b) treated with  5 mM Mg2+/1 mM EGTA; or (c) treated with 5 μM thapsigargin  for 30 min at 37°C. LFA-1 was detected with FITC-conjugated  LFA-1–specific mAb and analyzed by confocal microscopy. Red  color indicates fluorescence intensity exceeding a preset pixel  value (see Materials and Methods). This setting remained constant between samples. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Thapsigargin stimulates LFA-1 clustering. T cells adhered onto poly-L-lysine were (a) unstimulated; (b) treated with 5 mM Mg2+/1 mM EGTA; or (c) treated with 5 μM thapsigargin for 30 min at 37°C. LFA-1 was detected with FITC-conjugated LFA-1–specific mAb and analyzed by confocal microscopy. Red color indicates fluorescence intensity exceeding a preset pixel value (see Materials and Methods). This setting remained constant between samples. Bar, 10 μm.
Mentions: As the Ca2+ mobilizers did not cause a detectable increase in the affinity of LFA-1, we used confocal microscopy to investigate whether the membrane distribution of LFA-1 was altered in a manner facilitating adhesion. To analyze LFA-1 distribution, we highlighted the confocal microscopy images such that the membrane regions with the highest LFA-1 fluorescence (i.e., in the pixel intensity range 150–250, where 250 is the highest possible value) are depicted in red and all others in white (Fig. 4). Unstimulated cells (Fig. 4 a) and Mg2+/EGTA–stimulated cells (Fig. 4 b) have very little high intensity LFA-1 fluorescence (red) in comparison to that of thapsigargin-stimulated cells (Fig. 4 c). These observations were confirmed by quantifying (see Materials and Methods) the levels of fluorescence for each sample on regions of the membrane where there was no contact between cells (Table I, Free membrane). Statistical analysis (one way ANOVA) of these measurements confirmed that the level of fluorescence on the thapsigargin-stimulated cells, but not on the Mg2+/EGTA–stimulated T cells, was significantly increased compared to the level on the resting T cells (Table I, Significance levels). This increase in the intensity of LFA-1 fluorescence upon thapsigargin stimulation was detected with three distinct CD11a mAbs, 38, F110.22, and G25.2 (data not shown), and therefore does not represent the exposure of a particular epitope. These results suggested that thapsigargin might stimulate an increase in the cell surface expression of LFA-1. When measured by flow cytometry, however, results revealed that the level of LFA-1 expression after thapsigargin stimulation did not differ from LFA-1 expression on the resting or Mg2+/EGTA–treated cells (Table I, Flow cytometry). This suggested that the increase in signal strength observed by confocal microscopy upon thapsigargin stimulation reflects increased clustering that creates a higher LFA-1 fluorescence intensity. Similar results were obtained with the other Ca2+-mobilizing agents, with PdBu, and by XL-TCR–CD3 (data not shown).

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