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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.

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Migration of αMβ2 WT transfectants but not mock transfectants to Fg. Human 293 cells (5 × 105/well) expressing αMβ2 (WT, black bars) or mock-transfected cells containing only the neomycin resistance plasmid (white bars) were assessed for their ability to migrate to Fg (50 μg/ml, 150 nM), Fn (50 μg/ml), or medium alone placed in the lower wells of transwell plates. Some cells were pretreated with 20 μg/ml anti-β1 blocking mAb F4611 (A) or 100 nM NIF; M1/70 and 44a, 20 μg/ml αM-blocking mAb; IB4, 20 μg/ml β2-blocking mAb; or LM2/1, 20 μg/ml αM-nonblocking mAb (B) for 30 min before addition to the upper wells. Migration was assessed for 22 h at 37°C and migrated cells were fixed, stained, and counted. Migration data are expressed as mean cells/HPF ± SD for five random fields per well (A) or as a percentage of the migration of the WT cells to Fg (B) with duplicate wells in each experiment from three or more experiments. The x-axis indicates the addition to the upper wells containing cells over the addition to the lower wells. ***Medium alone.
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Figure 1: Migration of αMβ2 WT transfectants but not mock transfectants to Fg. Human 293 cells (5 × 105/well) expressing αMβ2 (WT, black bars) or mock-transfected cells containing only the neomycin resistance plasmid (white bars) were assessed for their ability to migrate to Fg (50 μg/ml, 150 nM), Fn (50 μg/ml), or medium alone placed in the lower wells of transwell plates. Some cells were pretreated with 20 μg/ml anti-β1 blocking mAb F4611 (A) or 100 nM NIF; M1/70 and 44a, 20 μg/ml αM-blocking mAb; IB4, 20 μg/ml β2-blocking mAb; or LM2/1, 20 μg/ml αM-nonblocking mAb (B) for 30 min before addition to the upper wells. Migration was assessed for 22 h at 37°C and migrated cells were fixed, stained, and counted. Migration data are expressed as mean cells/HPF ± SD for five random fields per well (A) or as a percentage of the migration of the WT cells to Fg (B) with duplicate wells in each experiment from three or more experiments. The x-axis indicates the addition to the upper wells containing cells over the addition to the lower wells. ***Medium alone.

Mentions: To investigate the relationship between αMβ2, Fg, and cell migration, we compared the ability of transfected cells expressing αMβ2 or mock-transfected cells to migrate toward Fg in a transwell system. Migration was allowed to proceed for 22 h at 37°C, at which time the cells adherent to the underside of the filter were fixed, stained, and counted. Cell viability, as judged by trypan blue exclusion, remained high (>95%) during the course of the assays. When the cells were placed in the upper chamber and a Fg concentration of 50 μg/ml in the lower chamber, a dramatic difference in the migration of the αMβ2 and mock-transfected cells was observed (Fig. 1 A). With the mock-transfected cells, 15 ± 3 cells migrated per HPF, whereas 396 ± 58 of the αMβ2-transfected cells migrated at the same time point. Background migration for each cell type was measured using medium alone in lower wells. Although the number of αMβ2-expressing cells recovered on the lower surface of the filter with only buffer present in the lower chamber was slightly higher (59 ± 9 cells/HPF) than for mock-transfected cells (15 ± 3 cells/HPF), the migration of the αMβ2 cells toward Fg was nearly sevenfold more than this background migration. Over the course of five experiments, the increase in migration of the αMβ2-transfected cells to Fg versus buffer was 671 ± 15%. Nevertheless, the mock-transfected cells were able to migrate as demonstrated when fibronectin (Fn) was placed in the lower wells. This migration of the mock-transfected cells to Fn was similar in extent to that of the αMβ2-transfected cells to Fg and was inhibited by an mAb (F4611) to the integrin β1 subunit (20 μg/ml), which had no effect on αMβ2 migration to Fg (see Fig. 1 A). These experiments were conducted in protein-free DMEM-F12, but similar results also were obtained in hybridoma serum-free medium.


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 transfectants but not mock transfectants to Fg. Human 293 cells (5 × 105/well) expressing αMβ2 (WT, black bars) or mock-transfected cells containing only the neomycin resistance plasmid (white bars) were assessed for their ability to migrate to Fg (50 μg/ml, 150 nM), Fn (50 μg/ml), or medium alone placed in the lower wells of transwell plates. Some cells were pretreated with 20 μg/ml anti-β1 blocking mAb F4611 (A) or 100 nM NIF; M1/70 and 44a, 20 μg/ml αM-blocking mAb; IB4, 20 μg/ml β2-blocking mAb; or LM2/1, 20 μg/ml αM-nonblocking mAb (B) for 30 min before addition to the upper wells. Migration was assessed for 22 h at 37°C and migrated cells were fixed, stained, and counted. Migration data are expressed as mean cells/HPF ± SD for five random fields per well (A) or as a percentage of the migration of the WT cells to Fg (B) with duplicate wells in each experiment from three or more experiments. The x-axis indicates the addition to the upper wells containing cells over the addition to the lower wells. ***Medium alone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193326&req=5

Figure 1: Migration of αMβ2 WT transfectants but not mock transfectants to Fg. Human 293 cells (5 × 105/well) expressing αMβ2 (WT, black bars) or mock-transfected cells containing only the neomycin resistance plasmid (white bars) were assessed for their ability to migrate to Fg (50 μg/ml, 150 nM), Fn (50 μg/ml), or medium alone placed in the lower wells of transwell plates. Some cells were pretreated with 20 μg/ml anti-β1 blocking mAb F4611 (A) or 100 nM NIF; M1/70 and 44a, 20 μg/ml αM-blocking mAb; IB4, 20 μg/ml β2-blocking mAb; or LM2/1, 20 μg/ml αM-nonblocking mAb (B) for 30 min before addition to the upper wells. Migration was assessed for 22 h at 37°C and migrated cells were fixed, stained, and counted. Migration data are expressed as mean cells/HPF ± SD for five random fields per well (A) or as a percentage of the migration of the WT cells to Fg (B) with duplicate wells in each experiment from three or more experiments. The x-axis indicates the addition to the upper wells containing cells over the addition to the lower wells. ***Medium alone.
Mentions: To investigate the relationship between αMβ2, Fg, and cell migration, we compared the ability of transfected cells expressing αMβ2 or mock-transfected cells to migrate toward Fg in a transwell system. Migration was allowed to proceed for 22 h at 37°C, at which time the cells adherent to the underside of the filter were fixed, stained, and counted. Cell viability, as judged by trypan blue exclusion, remained high (>95%) during the course of the assays. When the cells were placed in the upper chamber and a Fg concentration of 50 μg/ml in the lower chamber, a dramatic difference in the migration of the αMβ2 and mock-transfected cells was observed (Fig. 1 A). With the mock-transfected cells, 15 ± 3 cells migrated per HPF, whereas 396 ± 58 of the αMβ2-transfected cells migrated at the same time point. Background migration for each cell type was measured using medium alone in lower wells. Although the number of αMβ2-expressing cells recovered on the lower surface of the filter with only buffer present in the lower chamber was slightly higher (59 ± 9 cells/HPF) than for mock-transfected cells (15 ± 3 cells/HPF), the migration of the αMβ2 cells toward Fg was nearly sevenfold more than this background migration. Over the course of five experiments, the increase in migration of the αMβ2-transfected cells to Fg versus buffer was 671 ± 15%. Nevertheless, the mock-transfected cells were able to migrate as demonstrated when fibronectin (Fn) was placed in the lower wells. This migration of the mock-transfected cells to Fn was similar in extent to that of the αMβ2-transfected cells to Fg and was inhibited by an mAb (F4611) to the integrin β1 subunit (20 μg/ml), which had no effect on αMβ2 migration to Fg (see Fig. 1 A). These experiments were conducted in protein-free DMEM-F12, but similar results also were obtained in hybridoma serum-free medium.

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