Limits...
Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides.

Forsyth CB, Solovjov DA, Ugarova TP, Plow EF - J. Exp. Med. (2001)

Bottom Line: Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration.The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide.Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

View Article: PubMed Central - PubMed

Affiliation: Joseph J. Jacobs Center for Thrombosis and Vascular Biology, and the Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

ABSTRACT
Leukocyte migration is the hallmark of inflammation, and integrin alpha(M)beta(2) and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Show MeSH

Related in: MedlinePlus

Migration of αMβ2 WT cells to Fg is inhibited by P1 but stimulated by P2. Migration of αMβ2 WT cells was assessed to various combinations of optimal concentrations of 50 μg/ml Fg, 6 μM P1 peptide, 6 μM P2 peptide, or 10 nM D100 fragment in the upper or lower transwells as indicated. Fg or peptides added to the upper well were preincubated with cells for 30 min before addition of the entire mixture to the transwell. Data are means of cells per HPF with duplicate wells for each experiment from three or more experiments and expressed as percentage of WT ± SD.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193326&req=5

Figure 5: Migration of αMβ2 WT cells to Fg is inhibited by P1 but stimulated by P2. Migration of αMβ2 WT cells was assessed to various combinations of optimal concentrations of 50 μg/ml Fg, 6 μM P1 peptide, 6 μM P2 peptide, or 10 nM D100 fragment in the upper or lower transwells as indicated. Fg or peptides added to the upper well were preincubated with cells for 30 min before addition of the entire mixture to the transwell. Data are means of cells per HPF with duplicate wells for each experiment from three or more experiments and expressed as percentage of WT ± SD.

Mentions: To further examine the migratory activity of these Fg peptides, their ability to inhibit migration to Fg and each other was assessed (Fig. 5). When placed only in the upper well together with the cells, neither of the two peptides or the D100 fragment stimulated spontaneous migration of the αMβ2 cells, suggesting that the induction of cell migration to these derivatives was a chemotactic response. Furthermore, when placed in equal amounts in both the upper and lower wells, P1, P2, and the D100 fragment inhibited migration to itself, consistent with a chemotactic activity (data not shown). When testing the effects of the Fg derivatives in influencing cell migration to each other, some unexpected results were encountered. As anticipated, when the P1 peptide was added to the αMβ2-transfected cells in the upper chamber, it did inhibit migration to P2 and Fg in the lower chamber. In contrast, when P2 was added to the cells in the upper well, it stimulated rather than inhibited migration to P1 in the lower well (Fig. 5). To further explore the stimulatory effect of P2, its influence on αMβ2-mediated cell migration to Fg was assessed. As shown in Fig. 5, when added to the cells, P2 also stimulated migration to Fg. At a 6 μM concentration, P2 increased migration to Fg by 50% (relative to the migration of the cells to Fg alone, 100%). In contrast, both D100 (10 nM, 82% inhibition) and P1 (6 μM, 77% inhibition) inhibited migration of the αMβ2 cells under the same conditions. The Fg γ400–411 peptide, reported by other investigators to inhibit αMβ2 adhesion 50 had no effect on WT migration to Fg in concentrations as high as 50 μM (data not shown). This stimulatory effect of P2 was similar to that induced by two known activators of αMβ2. As shown in Fig. 6, 10 nM PMA and 2 μg/ml β-glucan were found to stimulate migration by 25 and 70%, respectively. The increases in migration induced by all three activators were statistically significant (P < 0.05) relative to the migration to Fg in their absence. Collectively, these data demonstrate a novel role for the P2 peptide in stimulating αMβ2-mediated migration to Fg.


Integrin alpha(M)beta(2)-mediated cell migration to fibrinogen and its recognition peptides.

Forsyth CB, Solovjov DA, Ugarova TP, Plow EF - J. Exp. Med. (2001)

Migration of αMβ2 WT cells to Fg is inhibited by P1 but stimulated by P2. Migration of αMβ2 WT cells was assessed to various combinations of optimal concentrations of 50 μg/ml Fg, 6 μM P1 peptide, 6 μM P2 peptide, or 10 nM D100 fragment in the upper or lower transwells as indicated. Fg or peptides added to the upper well were preincubated with cells for 30 min before addition of the entire mixture to the transwell. Data are means of cells per HPF with duplicate wells for each experiment from three or more experiments and expressed as percentage of WT ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193326&req=5

Figure 5: Migration of αMβ2 WT cells to Fg is inhibited by P1 but stimulated by P2. Migration of αMβ2 WT cells was assessed to various combinations of optimal concentrations of 50 μg/ml Fg, 6 μM P1 peptide, 6 μM P2 peptide, or 10 nM D100 fragment in the upper or lower transwells as indicated. Fg or peptides added to the upper well were preincubated with cells for 30 min before addition of the entire mixture to the transwell. Data are means of cells per HPF with duplicate wells for each experiment from three or more experiments and expressed as percentage of WT ± SD.
Mentions: To further examine the migratory activity of these Fg peptides, their ability to inhibit migration to Fg and each other was assessed (Fig. 5). When placed only in the upper well together with the cells, neither of the two peptides or the D100 fragment stimulated spontaneous migration of the αMβ2 cells, suggesting that the induction of cell migration to these derivatives was a chemotactic response. Furthermore, when placed in equal amounts in both the upper and lower wells, P1, P2, and the D100 fragment inhibited migration to itself, consistent with a chemotactic activity (data not shown). When testing the effects of the Fg derivatives in influencing cell migration to each other, some unexpected results were encountered. As anticipated, when the P1 peptide was added to the αMβ2-transfected cells in the upper chamber, it did inhibit migration to P2 and Fg in the lower chamber. In contrast, when P2 was added to the cells in the upper well, it stimulated rather than inhibited migration to P1 in the lower well (Fig. 5). To further explore the stimulatory effect of P2, its influence on αMβ2-mediated cell migration to Fg was assessed. As shown in Fig. 5, when added to the cells, P2 also stimulated migration to Fg. At a 6 μM concentration, P2 increased migration to Fg by 50% (relative to the migration of the cells to Fg alone, 100%). In contrast, both D100 (10 nM, 82% inhibition) and P1 (6 μM, 77% inhibition) inhibited migration of the αMβ2 cells under the same conditions. The Fg γ400–411 peptide, reported by other investigators to inhibit αMβ2 adhesion 50 had no effect on WT migration to Fg in concentrations as high as 50 μM (data not shown). This stimulatory effect of P2 was similar to that induced by two known activators of αMβ2. As shown in Fig. 6, 10 nM PMA and 2 μg/ml β-glucan were found to stimulate migration by 25 and 70%, respectively. The increases in migration induced by all three activators were statistically significant (P < 0.05) relative to the migration to Fg in their absence. Collectively, these data demonstrate a novel role for the P2 peptide in stimulating αMβ2-mediated migration to Fg.

Bottom Line: Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration.The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide.Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

View Article: PubMed Central - PubMed

Affiliation: Joseph J. Jacobs Center for Thrombosis and Vascular Biology, and the Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

ABSTRACT
Leukocyte migration is the hallmark of inflammation, and integrin alpha(M)beta(2) and its ligand fibrinogen (Fg) are key participants in this cellular response. Cells expressing wild-type or mutant alpha(M)beta(2) and Fg or its derivatives have been used to dissect the molecular requirements for this receptor-ligand pair to mediate cell migration. The major conclusions are that (a) Fg, its D fragment, and its P1 and P2 alpha(M)beta(2) recognition peptides support a chemotactic response; (b) when the I domain of alpha(L) was replaced with the I domain of alpha(M), the chimeric receptor supported cell migration to Fg; however, the alpha(M) subunit, containing the I domain but lacking the beta(2) subunit, supported migration poorly, thus, the alpha(M)I domain is necessary but not sufficient to support chemotaxis, and efficient migration requires the beta(2) subunit and alpha(M)I domain; and (c) in addition to supporting cell migration, P2 enhanced alpha(M)beta(2)-mediated chemotaxis to Fg and the P1 peptide. This activation was associated with exposure of the activation-dependent epitope recognized by monoclonal antibody 7E3 and was observed also with human neutrophils. Taken together, these data define specific molecular requirements for alpha(M)beta(2) to mediate cell migration to Fg derivatives and assign a novel proinflammatory activity to the P2 peptide.

Show MeSH
Related in: MedlinePlus