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Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization.

Chigaev A, Smagley Y, Sklar LA - BMC Immunol. (2014)

Bottom Line: Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation.We conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand.We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and University of New Mexico Cancer Center, Albuquerque 87131, NM, USA. achigaev@salud.unm.edu.

ABSTRACT

Background: Carbon monoxide (CO), a byproduct of heme degradation, is attracting growing attention from the scientific community. At physiological concentrations, CO plays a role as a signal messenger that regulates a number of physiological processes. CO releasing molecules are under evaluation in preclinical models for the management of inflammation, sepsis, ischemia/reperfusion injury, and organ transplantation. Because of our discovery that nitric oxide signaling actively down-regulates integrin affinity and cell adhesion, and the similarity between nitric oxide and CO-dependent signaling, we studied the effects of CO on integrin signaling and cell adhesion.

Results: We used a cell permeable CO releasing molecule (CORM-2) to elevate intracellular CO, and a fluorescent Very Late Antigen-4 (VLA-4, α4β1-integrin)-specific ligand to evaluate the integrin state in real-time on live cells. We show that the binding of the ligand can be rapidly down-modulated in resting cells and after inside-out activation through several Gαi-coupled receptors. Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation. Inhibition of VLA-4 ligand binding by CO had a dramatic effect on cell-cell interaction in a VLA-4/VCAM-1-dependent cell adhesion system.

Conclusions: We conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand. We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.

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Effect of CO donor on binding and dissociation of the LDV-FITC probe on resting and activated cells. LDV-FITC binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant FPR ΔST plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by addition of excess unlabeled competitor (LDV), and therefore, no autofluorescence can be seen. A. The experiment involved sequential additions of the LDV-FITC, and different concentrations of CORM-2 or vehicle. The non-specific binding of the probe was determined using LDV. Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves to the single exponential equation. B. The span of the single exponential fits for the dissociation curves (from A after LDV addition) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The sigmoidal dose–response (Hill slope = 1) was fit using GraphPad Prism. C. The sequential addition of the LDV-FITC, the high affinity FPR ligand (fMLFF), CORM-2 or vehicle, and LDV. LDV-FITC koffs were determined as described for A. The level of LDV-FITC binding corresponding to resting cells is indicated by the dashed line. D. The span of single exponential fits for the curves (from panel C) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The dose–response was fit analogously to B. E. The experiment involved addition of the LDV-FITC, RuCl3 or vehicle. F. The sequential addition of the LDV-FITC, and CORM-2. The “old” CORM-2 was prepared by incubating the solution for 48 hours at room temperature. The non-specific binding of the LDV-FITC probe was determined using LDV. For panels A, C, E, and F, a representative experiment of two independent experiments is shown.
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Fig2: Effect of CO donor on binding and dissociation of the LDV-FITC probe on resting and activated cells. LDV-FITC binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant FPR ΔST plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by addition of excess unlabeled competitor (LDV), and therefore, no autofluorescence can be seen. A. The experiment involved sequential additions of the LDV-FITC, and different concentrations of CORM-2 or vehicle. The non-specific binding of the probe was determined using LDV. Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves to the single exponential equation. B. The span of the single exponential fits for the dissociation curves (from A after LDV addition) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The sigmoidal dose–response (Hill slope = 1) was fit using GraphPad Prism. C. The sequential addition of the LDV-FITC, the high affinity FPR ligand (fMLFF), CORM-2 or vehicle, and LDV. LDV-FITC koffs were determined as described for A. The level of LDV-FITC binding corresponding to resting cells is indicated by the dashed line. D. The span of single exponential fits for the curves (from panel C) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The dose–response was fit analogously to B. E. The experiment involved addition of the LDV-FITC, RuCl3 or vehicle. F. The sequential addition of the LDV-FITC, and CORM-2. The “old” CORM-2 was prepared by incubating the solution for 48 hours at room temperature. The non-specific binding of the LDV-FITC probe was determined using LDV. For panels A, C, E, and F, a representative experiment of two independent experiments is shown.

Mentions: The VLA-4-specific ligand (LDV-FITC) is a small fluorescent probe based on the published structure of BIO1211, a CD49d/CD29 specific antagonist [30-32]. The molecule contains the Leu-Asp-Val (LDV) ligand binding motif from the alternatively spliced connecting segment-1 (CS-1) peptide of fibronectin. The major advantage of this probe is that it can be used to detect VLA-4 conformational changes on live cells in real-time in response to cell signaling [8,33,34]. The binding affinity detected using LDV-FITC varies in parallel with VCAM-1, the major natural VLA-4 ligand [35]. VCAM-1 contains the Ile-Asp-Ser (IDS) motif homologous to LDV, and VLA-4 interaction with VCAM-1 can be blocked by LDV-containing molecules [35-37]. To determine the effect of the CO donor on resting cells, samples were first treated with 25 nM LDV-FITC (Figure 2A). This concentration is about 2 fold higher than the dissociation constant for LDV-FITC binding to U937 cells without activation (Kd ~12 nM, [30]). Therefore, 70–80% of low affinity sites are occupied. Next, the addition of CORM-2 resulted in the dose-dependent dissociation of LDV-FITC that reached a steady-state 5–6 min after addition. Finally, an excess of unlabeled competitor (LDV) was added to determine the non-specific binding of the probe (Figure 2A). This induced rapid LDV-FITC dissociation with a rate (koff) similar to the rate reported for resting cells [35]. To determine the EC50 for the effect of CORM-2 on LDV-FITC binding, the span of the single exponential fits for the dissociation curves after LDV addition was plotted versus the logarithm of CORM-2 concentration (Figure 2B).Figure 2


Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization.

Chigaev A, Smagley Y, Sklar LA - BMC Immunol. (2014)

Effect of CO donor on binding and dissociation of the LDV-FITC probe on resting and activated cells. LDV-FITC binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant FPR ΔST plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by addition of excess unlabeled competitor (LDV), and therefore, no autofluorescence can be seen. A. The experiment involved sequential additions of the LDV-FITC, and different concentrations of CORM-2 or vehicle. The non-specific binding of the probe was determined using LDV. Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves to the single exponential equation. B. The span of the single exponential fits for the dissociation curves (from A after LDV addition) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The sigmoidal dose–response (Hill slope = 1) was fit using GraphPad Prism. C. The sequential addition of the LDV-FITC, the high affinity FPR ligand (fMLFF), CORM-2 or vehicle, and LDV. LDV-FITC koffs were determined as described for A. The level of LDV-FITC binding corresponding to resting cells is indicated by the dashed line. D. The span of single exponential fits for the curves (from panel C) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The dose–response was fit analogously to B. E. The experiment involved addition of the LDV-FITC, RuCl3 or vehicle. F. The sequential addition of the LDV-FITC, and CORM-2. The “old” CORM-2 was prepared by incubating the solution for 48 hours at room temperature. The non-specific binding of the LDV-FITC probe was determined using LDV. For panels A, C, E, and F, a representative experiment of two independent experiments is shown.
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Related In: Results  -  Collection

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Fig2: Effect of CO donor on binding and dissociation of the LDV-FITC probe on resting and activated cells. LDV-FITC binding and dissociation on U937 cells stably transfected with the non-desensitizing mutant FPR ΔST plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by addition of excess unlabeled competitor (LDV), and therefore, no autofluorescence can be seen. A. The experiment involved sequential additions of the LDV-FITC, and different concentrations of CORM-2 or vehicle. The non-specific binding of the probe was determined using LDV. Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves to the single exponential equation. B. The span of the single exponential fits for the dissociation curves (from A after LDV addition) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The sigmoidal dose–response (Hill slope = 1) was fit using GraphPad Prism. C. The sequential addition of the LDV-FITC, the high affinity FPR ligand (fMLFF), CORM-2 or vehicle, and LDV. LDV-FITC koffs were determined as described for A. The level of LDV-FITC binding corresponding to resting cells is indicated by the dashed line. D. The span of single exponential fits for the curves (from panel C) plotted versus logarithm of CORM-2 concentration. Means ± SEM of two independent determinations are shown. The dose–response was fit analogously to B. E. The experiment involved addition of the LDV-FITC, RuCl3 or vehicle. F. The sequential addition of the LDV-FITC, and CORM-2. The “old” CORM-2 was prepared by incubating the solution for 48 hours at room temperature. The non-specific binding of the LDV-FITC probe was determined using LDV. For panels A, C, E, and F, a representative experiment of two independent experiments is shown.
Mentions: The VLA-4-specific ligand (LDV-FITC) is a small fluorescent probe based on the published structure of BIO1211, a CD49d/CD29 specific antagonist [30-32]. The molecule contains the Leu-Asp-Val (LDV) ligand binding motif from the alternatively spliced connecting segment-1 (CS-1) peptide of fibronectin. The major advantage of this probe is that it can be used to detect VLA-4 conformational changes on live cells in real-time in response to cell signaling [8,33,34]. The binding affinity detected using LDV-FITC varies in parallel with VCAM-1, the major natural VLA-4 ligand [35]. VCAM-1 contains the Ile-Asp-Ser (IDS) motif homologous to LDV, and VLA-4 interaction with VCAM-1 can be blocked by LDV-containing molecules [35-37]. To determine the effect of the CO donor on resting cells, samples were first treated with 25 nM LDV-FITC (Figure 2A). This concentration is about 2 fold higher than the dissociation constant for LDV-FITC binding to U937 cells without activation (Kd ~12 nM, [30]). Therefore, 70–80% of low affinity sites are occupied. Next, the addition of CORM-2 resulted in the dose-dependent dissociation of LDV-FITC that reached a steady-state 5–6 min after addition. Finally, an excess of unlabeled competitor (LDV) was added to determine the non-specific binding of the probe (Figure 2A). This induced rapid LDV-FITC dissociation with a rate (koff) similar to the rate reported for resting cells [35]. To determine the EC50 for the effect of CORM-2 on LDV-FITC binding, the span of the single exponential fits for the dissociation curves after LDV addition was plotted versus the logarithm of CORM-2 concentration (Figure 2B).Figure 2

Bottom Line: Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation.We conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand.We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and University of New Mexico Cancer Center, Albuquerque 87131, NM, USA. achigaev@salud.unm.edu.

ABSTRACT

Background: Carbon monoxide (CO), a byproduct of heme degradation, is attracting growing attention from the scientific community. At physiological concentrations, CO plays a role as a signal messenger that regulates a number of physiological processes. CO releasing molecules are under evaluation in preclinical models for the management of inflammation, sepsis, ischemia/reperfusion injury, and organ transplantation. Because of our discovery that nitric oxide signaling actively down-regulates integrin affinity and cell adhesion, and the similarity between nitric oxide and CO-dependent signaling, we studied the effects of CO on integrin signaling and cell adhesion.

Results: We used a cell permeable CO releasing molecule (CORM-2) to elevate intracellular CO, and a fluorescent Very Late Antigen-4 (VLA-4, α4β1-integrin)-specific ligand to evaluate the integrin state in real-time on live cells. We show that the binding of the ligand can be rapidly down-modulated in resting cells and after inside-out activation through several Gαi-coupled receptors. Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation. Inhibition of VLA-4 ligand binding by CO had a dramatic effect on cell-cell interaction in a VLA-4/VCAM-1-dependent cell adhesion system.

Conclusions: We conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand. We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.

Show MeSH
Related in: MedlinePlus