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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 cell adhesion between U937 cells and VCAM-1-transfected B78H1 cells. A. Dot plot of flow cytometric analysis of cell aggregation. Cells were labeled with red and green fluorescent dyes. Next, cells were mixed at 0 time point. During data acquisition samples were maintained at 37°C, and continuously stirred with a magnetic stir bar. An increase in the number of aggregates was detected as green and red co-fluorescent particles indicated by the circular gate. B. Real-time cell aggregation plotted as % aggregates (Agg, %) versus time. The data were normalized to the non-specific aggregation determined as cell aggregation in the presence of excess unlabeled competitor (1 μM LDV). A representative experiment out of two experiments is shown. C. Statistical significance of the CO donor effect on cell aggregation. The aggregate percentage data from the last 5 min of the experiments (B) are compared using the unpaired t test. Means are significantly different (P < 0.05).
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Fig6: Effect of CO donor on cell adhesion between U937 cells and VCAM-1-transfected B78H1 cells. A. Dot plot of flow cytometric analysis of cell aggregation. Cells were labeled with red and green fluorescent dyes. Next, cells were mixed at 0 time point. During data acquisition samples were maintained at 37°C, and continuously stirred with a magnetic stir bar. An increase in the number of aggregates was detected as green and red co-fluorescent particles indicated by the circular gate. B. Real-time cell aggregation plotted as % aggregates (Agg, %) versus time. The data were normalized to the non-specific aggregation determined as cell aggregation in the presence of excess unlabeled competitor (1 μM LDV). A representative experiment out of two experiments is shown. C. Statistical significance of the CO donor effect on cell aggregation. The aggregate percentage data from the last 5 min of the experiments (B) are compared using the unpaired t test. Means are significantly different (P < 0.05).

Mentions: Next, to study the implications of the CO signaling pathway on integrin-dependent cell adhesion, we utilized a VLA-4/VCAM-1-specific real-time cell aggregation assay [35]. The specificity of cell aggregation in this model system was tested using anti-α4-integrin blocking mAb as well as unlabeled LDV that completely blocked cell aggregation [35,53]. Prior to mixing, U937 cells constitutively expressing VLA-4 were labeled with green fluorescent dye, and B78H1 cells stably transfected with human VCAM-1 were stained with red fluorescent dye (Figure 6A). The cell aggregates were detected as red and green co-fluorescent events in real-time. Cells were mixed and the baseline aggregation data were collected for the first three minutes. Next, the tube was removed and an aliquot of stock solution of carbon monoxide donor CORM-2 in DMSO or equal volume of vehicle (DMSO) were added. The tube was rapidly replaced and data acquisition was reestablished. The data were collected for up to 30 min (Figure 6B).Figure 6


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 cell adhesion between U937 cells and VCAM-1-transfected B78H1 cells. A. Dot plot of flow cytometric analysis of cell aggregation. Cells were labeled with red and green fluorescent dyes. Next, cells were mixed at 0 time point. During data acquisition samples were maintained at 37°C, and continuously stirred with a magnetic stir bar. An increase in the number of aggregates was detected as green and red co-fluorescent particles indicated by the circular gate. B. Real-time cell aggregation plotted as % aggregates (Agg, %) versus time. The data were normalized to the non-specific aggregation determined as cell aggregation in the presence of excess unlabeled competitor (1 μM LDV). A representative experiment out of two experiments is shown. C. Statistical significance of the CO donor effect on cell aggregation. The aggregate percentage data from the last 5 min of the experiments (B) are compared using the unpaired t test. Means are significantly different (P < 0.05).
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Related In: Results  -  Collection

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Fig6: Effect of CO donor on cell adhesion between U937 cells and VCAM-1-transfected B78H1 cells. A. Dot plot of flow cytometric analysis of cell aggregation. Cells were labeled with red and green fluorescent dyes. Next, cells were mixed at 0 time point. During data acquisition samples were maintained at 37°C, and continuously stirred with a magnetic stir bar. An increase in the number of aggregates was detected as green and red co-fluorescent particles indicated by the circular gate. B. Real-time cell aggregation plotted as % aggregates (Agg, %) versus time. The data were normalized to the non-specific aggregation determined as cell aggregation in the presence of excess unlabeled competitor (1 μM LDV). A representative experiment out of two experiments is shown. C. Statistical significance of the CO donor effect on cell aggregation. The aggregate percentage data from the last 5 min of the experiments (B) are compared using the unpaired t test. Means are significantly different (P < 0.05).
Mentions: Next, to study the implications of the CO signaling pathway on integrin-dependent cell adhesion, we utilized a VLA-4/VCAM-1-specific real-time cell aggregation assay [35]. The specificity of cell aggregation in this model system was tested using anti-α4-integrin blocking mAb as well as unlabeled LDV that completely blocked cell aggregation [35,53]. Prior to mixing, U937 cells constitutively expressing VLA-4 were labeled with green fluorescent dye, and B78H1 cells stably transfected with human VCAM-1 were stained with red fluorescent dye (Figure 6A). The cell aggregates were detected as red and green co-fluorescent events in real-time. Cells were mixed and the baseline aggregation data were collected for the first three minutes. Next, the tube was removed and an aliquot of stock solution of carbon monoxide donor CORM-2 in DMSO or equal volume of vehicle (DMSO) were added. The tube was rapidly replaced and data acquisition was reestablished. The data were collected for up to 30 min (Figure 6B).Figure 6

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