Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of CO donor on surface expression of VLA-4 (CD49d/CD29 heterodimer). U937 cells were treated with vehicle (DMSO, control), and CO donor (150 μM CORM-2) for 30 min at 37°C. Next, cells were placed on ice and stained with primary labeled anti-CD29 and anti-CD49d antibodies, or the isotype control. A. Histograms of anti-CD29 and anti-CD49d antibodies are shown in black, and the isotype control is grey. B. Bar graphs of mean channel fluorescence (MCF) ± SEM (n =5) for unstained cells (autofluorescence), nonspecific binding to cells (isotype control), cells treated with vehicle (DMSO), and cells treated with CORM-2 are shown (10,000 gated events for each sample were collected). One representative experiment of three experiments is shown. * indicates means are significantly different (P <0.05) as estimated by the unpaired t test analysis using GraphPad Prism software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4221689&req=5

Fig5: Effect of CO donor on surface expression of VLA-4 (CD49d/CD29 heterodimer). U937 cells were treated with vehicle (DMSO, control), and CO donor (150 μM CORM-2) for 30 min at 37°C. Next, cells were placed on ice and stained with primary labeled anti-CD29 and anti-CD49d antibodies, or the isotype control. A. Histograms of anti-CD29 and anti-CD49d antibodies are shown in black, and the isotype control is grey. B. Bar graphs of mean channel fluorescence (MCF) ± SEM (n =5) for unstained cells (autofluorescence), nonspecific binding to cells (isotype control), cells treated with vehicle (DMSO), and cells treated with CORM-2 are shown (10,000 gated events for each sample were collected). One representative experiment of three experiments is shown. * indicates means are significantly different (P <0.05) as estimated by the unpaired t test analysis using GraphPad Prism software.

Mentions: To study the effect of CO signaling on VLA-4 surface expression, cells were treated with the CO donor or vehicle for 30 min at 37°C. Next, cells were stained with primary labeled antibody against α4- and β1-integrin subunits (Figure 5). Analysis of antibody binding revealed a decrease in the surface expression of both integrin subunits that varied from 10% to 26% in multiple experiments. Given the small sample to sample variation this difference was statistically significant (Figure 5B). However, this difference in the expression of VLA-4 subunits detected after 30 min of CO donor treatment cannot account for rapid and dramatic decrease in the VLA-4-specific ligand binding that has been detected after CO donor addition (Figure 2B,C).Figure 5


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 surface expression of VLA-4 (CD49d/CD29 heterodimer). U937 cells were treated with vehicle (DMSO, control), and CO donor (150 μM CORM-2) for 30 min at 37°C. Next, cells were placed on ice and stained with primary labeled anti-CD29 and anti-CD49d antibodies, or the isotype control. A. Histograms of anti-CD29 and anti-CD49d antibodies are shown in black, and the isotype control is grey. B. Bar graphs of mean channel fluorescence (MCF) ± SEM (n =5) for unstained cells (autofluorescence), nonspecific binding to cells (isotype control), cells treated with vehicle (DMSO), and cells treated with CORM-2 are shown (10,000 gated events for each sample were collected). One representative experiment of three experiments is shown. * indicates means are significantly different (P <0.05) as estimated by the unpaired t test analysis using GraphPad Prism software.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4221689&req=5

Fig5: Effect of CO donor on surface expression of VLA-4 (CD49d/CD29 heterodimer). U937 cells were treated with vehicle (DMSO, control), and CO donor (150 μM CORM-2) for 30 min at 37°C. Next, cells were placed on ice and stained with primary labeled anti-CD29 and anti-CD49d antibodies, or the isotype control. A. Histograms of anti-CD29 and anti-CD49d antibodies are shown in black, and the isotype control is grey. B. Bar graphs of mean channel fluorescence (MCF) ± SEM (n =5) for unstained cells (autofluorescence), nonspecific binding to cells (isotype control), cells treated with vehicle (DMSO), and cells treated with CORM-2 are shown (10,000 gated events for each sample were collected). One representative experiment of three experiments is shown. * indicates means are significantly different (P <0.05) as estimated by the unpaired t test analysis using GraphPad Prism software.
Mentions: To study the effect of CO signaling on VLA-4 surface expression, cells were treated with the CO donor or vehicle for 30 min at 37°C. Next, cells were stained with primary labeled antibody against α4- and β1-integrin subunits (Figure 5). Analysis of antibody binding revealed a decrease in the surface expression of both integrin subunits that varied from 10% to 26% in multiple experiments. Given the small sample to sample variation this difference was statistically significant (Figure 5B). However, this difference in the expression of VLA-4 subunits detected after 30 min of CO donor treatment cannot account for rapid and dramatic decrease in the VLA-4-specific ligand binding that has been detected after CO donor addition (Figure 2B,C).Figure 5

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