(A) In a low fMLP concentration gradient, both GFP and Rac2-T17N pretreated human neutrophils showed normal migration tracks toward the source of fMLP (labeled with red asterisk), suggesting that the inhibition of Rac1 didn’t block neutrophil chemotaxis. However, Rac1-T17N pretreated human neutrophils showed impaired migration tracks toward the source of fMLP (labeled with red asterisk), indicating that the inhibition of Rac1 was able to block neutrophil chemotaxis in a low concentration gradient of fMLP. (B)In a high fMLP concentration gradient, both GFP-Tat and Rac1-T17N pretreated human neutrophils show normal cell tracks toward the source of fMLP (red asterisk), suggesting the inhibition of Rac1 didn’t block neutrophil chemotaxis. However, Rac2-T17N pretreated human neutrophils exhibited shorter migration tracks toward the source of fMLP (red asterisk), indicating the inhibition of Rac2 was able to block neutrophil chemotaxis in a high fMLP concentration gradient. (Results are representive from 3 independent experiments)

(A) Human neutrophils were stimulated with a low uniform concentration of fMLP. All the cell tracks are shown at the top left of panel A. The cell stack of sample cell 14 is shown at the top right of panel A. At the bottom of panel A, the morphological changes of sample cell 14 are shown in the form of a schematic cell, in which the green regions represent the expansion area of the cell and the red regions represent the retraction area of the cell. (B) Human neutrophils were stimulated in a high uniform concentration of fMLP. All the cell tracks are shown at the top left of panel B. The cell stack of sample cell 16 is shown at the top right of panel B. At the bottom of panel B, the morphological changes of sample cell 16 are shown schematically, as described above.

(A) Sequential DIC images of a wild type mouse neutrophil are shown during fMLP stimulation at the left of panel A. At the right of panel A, the measurements of cell area and centroid speed averaged from 30 cells were plotted against time. (B) Sequential DIC images of Rac1^−/− mouse neutrophils are shown during fMLP sitmulation at the left of panel B. At the right of panel B, the measurements of cell area and centroid speed averaged from 30 cells were plotted against time. (C) Sequential DIC images of Rac2^−/− mouse neutrophils are shown during fMLP stimulation at the left of panel C. At the right of panel C, the measurements of cell area and centroid speed averaged from 30 cells were plotted against time. (Results are collected from three independent experiments)

(A) Freshly prepared human neutrophils were allowed to adhere to fibronectin coated surface for 1 hour, were stimulated with the final concentration of 1×10⁻⁷ M fMLP for the time period indicated, and active Rac1 was detected by affinity-based PBD pulldown assay as described in Materials and Methods.. (B) Adherent human neutrophils were stimulated by 1×10⁻⁷ M fMLP for the times indicated. Active Rac2 was detected by affinity-based PBD pulldown assay. (C) The ratio of active Rac at each time point as compared to time zero was described as [Active Rac (time X)/Total Rac (time X)]/[Active Rac (0 min)/Total Rac (0 min)]. Therefore, if the ratio of active Rac to the zero time is 1, it means no activation of Rac. If the ratio is more than 1, it means activation of Rac, and if the ratio is less than 1, it means inhibition of Rac activation. The activation of Rac1 was increased throughout the first 4 minutes of stimulation, then dropped sharply between 4–6 minutes, but increased again after 7 minutes. Conversely, the activation of Rac2 was increased throughout the first 7 minutes, then decreased sharply after this time. (The result is representive of two independent experiments)

(A) Human neutrophils were stimulated in a low concentration fMLP gradient. All the cell tracks are shown at the top left of panel A and the gradient source was indicated by the red asterisk. The cell stack of sample cell 11 is shown at the top right of panel A. At the bottom of panel A, the morphological changes of sample cell 11 at various times are shown in the form of a schematic cell, in which the green regions represent the expansion area of the cell and the red regions represent the retraction area of the cell. (B) Human neutrophils were stimulated in a high concentration fMLP gradient. All the cell tracks are shown at the top left of panel B and the gradient source was indicated by the red asterisk. The cell stack of sample cell 12 is shown at the top right of panel B. At the bottom of panel B, the morphological changes of sample cell 12 are shown in the form of schematic cells, as described above.

(A) Left- Sequential DIC images of an untreated human neutrophil are shown during stimulation with uniform 10⁻⁷ M fMLP. Right- the measurements of cell area and centroid speed averaged from 30 cells were plotted against the time, where the time of fMLP addition (zero) is labeled with a red asterisk on the X-axis. (B) Sequential DIC images of a Rac1-T17N pretreated human neutrophil are shown during stimulation with uniform 10⁻⁷ M fMLP (left side of panel B). At the right side of panel B, the measurements of cell area and centroid speed averaged from 30 cells were plotted against the time, where the time of fMLP addition (zero) is labeled with a red asterisk on the X-axis. (C) Sequential DIC images of a Rac2-T17N pretreated human neutrophil are shown during a uniform 10⁻⁷ M fMLP stimulation (left side of panel C). At the right side of panel C, the measurements of cell area and centroid speed averaged from 30 cells are plotted against the time, where the time of fMLP addition (zero) is labeled with a red asterisk on the X-axis. (Results are collected from three independent experiments)

(A) Freshly prepared human neutrophils were allowed to adhere to fibronectin-coated surface for 1 hour, stimulated with the final concentration of 1×10⁻⁷ M fMLP, and immunostained for Rac1 (green) and F-actin (red) at 0 min, 0.5 min, 1 min, 3 min, 5 min, 7 min and 9 min with Rac1-specific antibody (Upstate, 23A8) and phalloidin. (B) Freshly prepared human neutrophils were allowed adhere to fibronectin-coated surface for 1 hour, stimulated with the final concentration of 1×10⁻⁷ M fMLP, and immunostained for Rac2 (green) and F-actin (red) at 0 min, 0.5 min, 1 min, 3 min, 5 min, 7 min and 9 min with Rac2-specific antibody (R786) and phalloidin. In both A and B, the morphological changes were monitored through the corresponding DIC-DAPI images (top panels). (The result is representive of two independent experiment)

In this model, the X axis represents the distance from the emigration sites, where neutrophils migrate out of the blood vessels, to the infectious sites, where neutrophils exhibit inflammatory responses, such as superoxide production. The Y axis represents the predicted and deduced concentration range of fMLP (in log scale) over the distance from the emigration sites, where the fMLP concentration is lowest (1×10-9 M) to the infectious sites, where the fMLP concentration is highest (1×10-6 M). At the emigration site where the concentration of fMLP is low, neutrophils will activate Rac1 as the predominant form of Rac for initiating chemotaxis and directional migration. As neutrophils move towards the infectious sites, they experience a higher concentration of fMLP and activate both Rac1 and Rac2 to support both chemotaxis and inflammatory activity, such as superoxide production. Once neutrophils eventually reach the infectious site, under the high concentration of fMLP, only Rac2 is activated and the cells stop moving, allowing the neutrophils to stay at the infectious site for performing their inflammatory functions.