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First published online 25 October 2005
doi: 10.1242/jcs.02632

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Fundamentally different roles for LFA-1, Mac-1 and ₄-integrin in neutrophil chemotaxis

Immunology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, Alberta T2N 4N1, Canada

View larger version (72K): [in a new window]   Fig. 1. Analysis of cell movement. (A) Region of under agarose assay that is analyzed during the cell tracking experiments. The dotted box represents the region being recorded, which is located adjacent to the neutrophil-containing well. (B) Example of how a cell is tracked in the cell tracking assay. The neutrophil-containing well is visible on the left, and the cells are migrating towards a source of fMLP located off-screen to the right. The path taken by the cell is indicated by the white, curved line. The distance each cell moves between each time point (dotted line) is calculated, and then the time points are summed to give the total distance moved by the cell (dist). Next, the total distance the cell migrates towards the chemoattractant was calculated. This value is equal to the displacement of the cell along the x-axis (x). Speed was calculated by dividing dist by the duration of the experiment. Chemotactic index (C.I.) was calculated by dividing x by dist.

View larger version (20K): [in a new window]   Fig. 2. Random migration (chemokinesis) of neutrophils in uniform concentrations of fMLP and IL-8. Cells are migrating on substrata consisting of serum protein (serum), fibrinogen (Fbgn), fibrinogen and ICAM-1 (Fbgn+ICAM), or fibronectin (Fn). (A) Chemotactic index of cells migrating in uniform concentrations of fMLP and IL-8. (B) Speed of migration in uniform concentrations of fMLP and IL-8. (C) Tracks plotting the migration of ten representative cells in uniform concentrations of fMLP (left) and IL-8 (right). Scale is in µm. Results are shown as mean±s.e.m., n=5. No statistical differences were observed (Student's t-test).

View larger version (24K): [in a new window]   Fig. 3. Chemotaxis of neutrophils to fMLP, IL-8, and in the hierarchy on substrata of serum protein (serum) or fibrinogen (Fbgn). (A) Effect of different substrata on the number of cells migrating towards (white bars) and away (black bars) from fMLP and IL-8. (B) Chemotactic index (C.I.) of cells migrating to fMLP and IL-8. (C) Speed of migration to fMLP and IL-8. (D) Tracks plotting the migration of ten representative cells to fMLP on serum and fibrinogen. (E) Tracks plotting the migration of ten representative cells to IL-8 on serum and fibrinogen. Scale is in µm. (F) Patterns of cell migration in competing gradients of fMLP and IL-8. The number of cells migrating to fMLP and IL-8 (x-axis), as well as the number of cells migrating perpendicular to the chemoattractants (y-axis) are shown. Results are shown as mean±s.e.m., n=6 for panels A,D,E,F; n=4 for panels B,C. *P<0.05 compared with migration on serum protein.

View larger version (25K): [in a new window]   Fig. 4. Chemotaxis of neutrophils to fMLP, IL-8 and in the hierarchy on substrata of serum protein (serum), fibrinogen (Fbgn), or fibrinogen + ICAM-1 (Fbgn + ICAM-1). (A) Effect of different substrata on the number of cells migrating towards (white bars) and away (black bars) from fMLP and IL-8. (B) Chemotactic index (C.I.) of cells migrating to fMLP and IL-8. (C) Speed of migration to fMLP and IL-8. (D) Tracks plotting the migration of ten representative cells to fMLP (left) or IL-8 (right) on fibronectin + ICAM-1. Scale is in µm. (E) Patterns of cell migration in competing gradients of fMLP and IL-8. The number of cells migrating to fMLP and IL-8 (x-axis), as well as the number of cells migrating perpendicular to the chemoattractants (y-axis) are shown. Results are shown as mean±s.e.m., n=6 for panels A,D,E; n=4 for panels B,C. *P<0.05 compared with migration on serum protein.

View larger version (19K): [in a new window]   Fig. 5. Chemotaxis of neutrophils to fMLP and IL-8 on VCAM-1. (A) Effect of different substrata on the number of cells migrating towards (white bars) and away (black bars) from fMLP and IL-8. (B) Chemotactic index (C.I.) of cells migrating to fMLP and IL-8. (C) Speed of migration to fMLP and IL-8. (D) Paths of ten representative cells migrating to fMLP (left) and IL-8 (right) on VCAM-1. Scale is in µm. Results are shown as mean±s.e.m., n=5 for panels A,D; n=3 for panels B,C. *P<0.05 compared to migration on serum protein.

View larger version (26K): [in a new window]   Fig. 6. Chemotaxis of neutrophils to fMLP, IL-8, and in the hierarchy on substrata of serum protein (serum) or fibronectin. (A) Effect of different substrata on the number of cells migrating towards (white bars) and away (black bars) from fMLP and IL-8. (B) Chemotactic index (C.I.) of cells migrating to fMLP and IL-8. (C) Speed of migration to fMLP and IL-8. (D) Tracks plotting the migration of ten representative cells to fMLP (left) or IL-8 (right) on fibronectin. Scale is in µm. (E) Patterns of cell migration in competing gradients of fMLP and IL-8. The number of cells migrating to fMLP and IL-8 (x-axis), as well as the number of cells migrating perpendicular to the chemoattractants (y-axis) are shown. (F) Chemotactic ratio of cells migrating to fMLP or IL-8 through transwell chambers coated with either serum protein (serum) or fibronectin. Results are shown as mean±s.e.m., n=5 for panels A,D; n=3 for panels B,C. *P<0.05 compared with migration on serum protein. P<0.05, compared with chemotaxis on membranes coated in serum. P<0.05, compared with chemokinesis on membranes coated in serum.

View larger version (27K): [in a new window]   Fig. 7. Effect of blocking antibodies against LFA-1, Mac-1 and 4-integrin on chemotaxis to end-target chemoattractants (fMLP, C5a) and to intermediary chemoattractant (IL-8, LTB4). Cells are chemotaxing on serum protein, which contain ligands for all three integrins. n=6, *P<0.05 compared with isotype.

View larger version (15K): [in a new window]   Fig. 8. Effect of blocking LFA-1 and 4-integrin on chemotaxis to fMLP. (A) Patterns of cell migration during chemotaxis to fMLP. (B) Chemotactic index (C.I.) of cells migrating to fMLP. (C) Speed of migration to fMLP. Results are shown as mean±s.e.m., n=3. *P<0.05 compared to isotype. P<0.05 compared with anti-4-integrin. P<0.05 compared with migration on serum protein away from fMLP.

View larger version (37K): [in a new window]   Fig. 9. Mac-1 activation on cells moving on serum towards single gradients of fMLP or IL-8. Cells are either uninhibited (UT) or treated with the p38 MAPK inhibitor SB203580. (A) Relative activation of Mac-1. Values are expressed as fold-activation, compared with fMLP, UT. (B) Total Mac-1 on cells, as measured using an activation-independent antibody. (C) DIC and fluorescent microscopy showing the localization of active Mac-1 on cells moving to fMLP, with and without p38 MAPK inhibition. (D) Effect of p38 MAPK inhibition on chemotaxis to fMLP and IL-8. Integrin data represents only the integrins in contact with the substratum, and are expressed as fluorescent intensity relative to fMLP, UT. Results are shown as mean±s.e.m., n=minimum of 5. *P<0.05 compared with untreated.

View larger version (40K): [in a new window]   Fig. 10. LFA-1 activation on cells moving on serum towards single gradients of fMLP or IL-8. (A) Relative activation of LFA-1. Values are expressed as fold-activation, compared with fMLP. (B) Total LFA-1 on cells, as measured using an activation-independent antibody. (C) DIC and fluorescent microscopy showing the localization of active LFA-1 on cells moving to fMLP (top) or IL-8 (bottom). Integrin data represent only the integrins in contact with the substratum, and is expressed as fluorescent intensity relative to fMLP. Results are shown as mean±s.e.m., n=minimum of 5. *P<0.05 compared with fMLP.

View larger version (27K): [in a new window]   Fig. 11. Integrin activation on cells migrating on serum in competing gradients of fMLP and IL-8. Cells are either uninhibited (UT) or treated with the p38 MAPK inhibitor SB203580. (A) Relative Mac-1 activation. Values are expressed as fold-activation, compared with UT. (B) Relative LFA-1 activation. Values are expressed as fold-activation, compared with UT. (C) Total Mac-1 and LFA-1 on cells, as measured using activation-independent antibodies. (D) Effect of blocking antibodies against Mac-1 and LFA-1 on chemotaxis in the hierarchy. (E) Effect of p38 MAPK inhibition on chemotaxis in the hierarchy, and the roles of LFA-1 and Mac-1 in mediating this chemotaxis. Integrin data represents only the integrins in contact with the substratum, and are expressed as fluorescent intensity relative to fMLP, UT. Results are shown as mean±s.e.m., n=minimum of 5. *P<0.05 compared to untreated.

View larger version (23K): [in a new window]   Fig. 12. Proposed signaling model. Chemotaxis involves three signals that, together, direct the cell towards a site of infection. The first of these signals is generated by p38 MAPK, and allows a cell to `ignore' intermediary chemoattractants (a). The second signal is an inside-out signal, generated by the chemoattractants, which results in the activation of the integrins (b). Lastly, a signal is generated by integrin-ligand interactions, and provides outside-in signals that are required to `steer' the cell (c). Lines with arrow heads at the ends represent stimulatory pathways. Lines with bars at the end represent inhibitory pathways.