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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 hemin on binding and dissociation of the LDV-FITC probe on resting and activated U937 cells. LDV-FITC probe binding and dissociation on U937 cells plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by the addition of excess unlabeled competitor (LDV 2 μM), and therefore, no cell autofluorescence can be seen. A. The experiment involved sequential addition of the fluorescent LDV-FITC probe (25 nM), and different concentrations of hemin (6–100 μM) or DMSO (vehicle). The non-specific binding of the LDV-FITC probe was determined using excess unlabeled competitor (LDV). Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves (after LDV addition) to the single exponential equation. The range of koff is shown. B. The span of the single exponential fits for the dissociation curves (from panel A after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit (Hill slope = 1) was obtained using GraphPad Prism software. C. The experiment was conducted using U937 cells stably transfected with the FPR ΔST receptor, and involved sequential addition of the fluorescent LDV-FITC probe (4 nM), the high affinity FPR ligand N-formyl-Met-Leu-Phe-Phe (100 nM), hemin (1.5-100 μM) or DMSO (control), and LDV (2 μM). LDV-FITC dissociation rates (koff) were determined as described for panel A. D. The span of the single exponential fits for the dissociation curves (from panel C, after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit was obtained analogously to panel B.
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Fig4: Effect of hemin on binding and dissociation of the LDV-FITC probe on resting and activated U937 cells. LDV-FITC probe binding and dissociation on U937 cells plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by the addition of excess unlabeled competitor (LDV 2 μM), and therefore, no cell autofluorescence can be seen. A. The experiment involved sequential addition of the fluorescent LDV-FITC probe (25 nM), and different concentrations of hemin (6–100 μM) or DMSO (vehicle). The non-specific binding of the LDV-FITC probe was determined using excess unlabeled competitor (LDV). Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves (after LDV addition) to the single exponential equation. The range of koff is shown. B. The span of the single exponential fits for the dissociation curves (from panel A after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit (Hill slope = 1) was obtained using GraphPad Prism software. C. The experiment was conducted using U937 cells stably transfected with the FPR ΔST receptor, and involved sequential addition of the fluorescent LDV-FITC probe (4 nM), the high affinity FPR ligand N-formyl-Met-Leu-Phe-Phe (100 nM), hemin (1.5-100 μM) or DMSO (control), and LDV (2 μM). LDV-FITC dissociation rates (koff) were determined as described for panel A. D. The span of the single exponential fits for the dissociation curves (from panel C, after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit was obtained analogously to panel B.

Mentions: Heme oxygenase is expressed in U937 cells [49-51], and exogenous hemin can be used to modulate cell signaling in this cell line [27,52]. To study the effect of hemin on LDV-FITC binding on resting and activated cells, experiments were conducted in a manner similar to the CO donor experiments (Figure 2). Resting U937 cells were treated with 25 nM LDV-FITC (Figure 4A). Next, appropriate concentrations of hemin were added. We observed a slow dose-dependent decrease in the LDV-FITC signal. Finally, to determine the non-specific binding of the LDV-FITC probe, excess unlabeled competitor was added 10–12 min later. As in the case of the CO donor, the ligand dissociation rate (koff) was similar to the rate reported for resting cells. To determine the EC50 for the effect of hemin on LDV-FITC binding, the span of single exponential fits for the dissociation curves after LDV addition was plotted versus the logarithm of hemin concentration (Figure 4B). The effect of hemin was similar to the effect of the CO donor on resting cells.Figure 4


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 hemin on binding and dissociation of the LDV-FITC probe on resting and activated U937 cells. LDV-FITC probe binding and dissociation on U937 cells plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by the addition of excess unlabeled competitor (LDV 2 μM), and therefore, no cell autofluorescence can be seen. A. The experiment involved sequential addition of the fluorescent LDV-FITC probe (25 nM), and different concentrations of hemin (6–100 μM) or DMSO (vehicle). The non-specific binding of the LDV-FITC probe was determined using excess unlabeled competitor (LDV). Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves (after LDV addition) to the single exponential equation. The range of koff is shown. B. The span of the single exponential fits for the dissociation curves (from panel A after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit (Hill slope = 1) was obtained using GraphPad Prism software. C. The experiment was conducted using U937 cells stably transfected with the FPR ΔST receptor, and involved sequential addition of the fluorescent LDV-FITC probe (4 nM), the high affinity FPR ligand N-formyl-Met-Leu-Phe-Phe (100 nM), hemin (1.5-100 μM) or DMSO (control), and LDV (2 μM). LDV-FITC dissociation rates (koff) were determined as described for panel A. D. The span of the single exponential fits for the dissociation curves (from panel C, after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit was obtained analogously to panel B.
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Related In: Results  -  Collection

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Fig4: Effect of hemin on binding and dissociation of the LDV-FITC probe on resting and activated U937 cells. LDV-FITC probe binding and dissociation on U937 cells plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by the addition of excess unlabeled competitor (LDV 2 μM), and therefore, no cell autofluorescence can be seen. A. The experiment involved sequential addition of the fluorescent LDV-FITC probe (25 nM), and different concentrations of hemin (6–100 μM) or DMSO (vehicle). The non-specific binding of the LDV-FITC probe was determined using excess unlabeled competitor (LDV). Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves (after LDV addition) to the single exponential equation. The range of koff is shown. B. The span of the single exponential fits for the dissociation curves (from panel A after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit (Hill slope = 1) was obtained using GraphPad Prism software. C. The experiment was conducted using U937 cells stably transfected with the FPR ΔST receptor, and involved sequential addition of the fluorescent LDV-FITC probe (4 nM), the high affinity FPR ligand N-formyl-Met-Leu-Phe-Phe (100 nM), hemin (1.5-100 μM) or DMSO (control), and LDV (2 μM). LDV-FITC dissociation rates (koff) were determined as described for panel A. D. The span of the single exponential fits for the dissociation curves (from panel C, after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose–response fit was obtained analogously to panel B.
Mentions: Heme oxygenase is expressed in U937 cells [49-51], and exogenous hemin can be used to modulate cell signaling in this cell line [27,52]. To study the effect of hemin on LDV-FITC binding on resting and activated cells, experiments were conducted in a manner similar to the CO donor experiments (Figure 2). Resting U937 cells were treated with 25 nM LDV-FITC (Figure 4A). Next, appropriate concentrations of hemin were added. We observed a slow dose-dependent decrease in the LDV-FITC signal. Finally, to determine the non-specific binding of the LDV-FITC probe, excess unlabeled competitor was added 10–12 min later. As in the case of the CO donor, the ligand dissociation rate (koff) was similar to the rate reported for resting cells. To determine the EC50 for the effect of hemin on LDV-FITC binding, the span of single exponential fits for the dissociation curves after LDV addition was plotted versus the logarithm of hemin concentration (Figure 4B). The effect of hemin was similar to the effect of the CO donor on resting cells.Figure 4

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