The use of lymphocyte function-associated antigen (LFA)-1-deficient mice to determine the role of LFA-1, Mac-1, and alpha4 integrin in the inflammatory response of neutrophils

Abstract

After injury or infection, neutrophils rapidly migrate from the circulation into tissues by means of an orderly progression of adhesion receptor engagements. Neutrophils have been previously considered to use selectins exclusively to roll on vessels before an adhesion step mediated by the beta2 integrins, lymphocyte function-associated antigen (LFA)-1, and Mac-1. Here we use LFA-1(-/-) mice, function blocking monoclonal antibodies, and intravital microscopy to investigate the roles of LFA-1, Mac-1, and alpha4 integrins in neutrophil recruitment in vivo. For the first time, we show that LFA-1 makes a contribution to neutrophil rolling by stabilizing the transient attachment or tethering phase of rolling. In contrast, Mac-1 does not appear to be important for either rolling or firm adhesion, but instead contributes to emigration from the vessel. Blocking Mac-1 in the presence of LFA-1 significantly reduces emigration, suggesting cooperation between these two integrins. Low levels of alpha4beta1 integrin can be detected on neutrophils from LFA-1(+/+) and (-/-) mice. These cells make use of alpha4beta1 during the rolling phase, particularly in the absence of LFA-1. Thus LFA-1 and alpha4beta1, together with the selectins, are involved in the rolling phase of neutrophil recruitment, and, in turn, affect the later stages of the transmigration event.

Figure 1

The migration of neutrophils at 4 h after TG treatment into the peritoneum of LFA-1^+/+ and ^−/− mice in the presence of mAbs specific for LFA-1, Mac-1, and α4 integrins either singly or in combination. For LFA-1^+/+ mice, anti–LFA-1 and anti–Mac-1 mAbs, when administered alone, significantly inhibit neutrophil migration. All mAb pairs were significantly effective in inhibiting migration compared with the control. In addition, all pairs of mAbs block significantly more than single mAbs (P < 0.001), indicating that α4 integrin cooperates with LFA-1 and Mac-1 in the migration of LFA-1^+/+ neutrophils. For LFA-1^−/− mice, only anti-α4 integrin inhibits neutrophil migration as a single mAb and does not inhibit more significantly when in combination with anti–Mac-1 mAb (α4 versus α4/Mac-1 mAbs; P > 0.05). These data are representative of three complete experiments which utilized 3–10 mice per condition per experiment. Data are reported as mean ± SEM.

Figure 1

The migration of neutrophils at 4 h after TG treatment into the peritoneum of LFA-1^+/+ and ^−/− mice in the presence of mAbs specific for LFA-1, Mac-1, and α4 integrins either singly or in combination. For LFA-1^+/+ mice, anti–LFA-1 and anti–Mac-1 mAbs, when administered alone, significantly inhibit neutrophil migration. All mAb pairs were significantly effective in inhibiting migration compared with the control. In addition, all pairs of mAbs block significantly more than single mAbs (P < 0.001), indicating that α4 integrin cooperates with LFA-1 and Mac-1 in the migration of LFA-1^+/+ neutrophils. For LFA-1^−/− mice, only anti-α4 integrin inhibits neutrophil migration as a single mAb and does not inhibit more significantly when in combination with anti–Mac-1 mAb (α4 versus α4/Mac-1 mAbs; P > 0.05). These data are representative of three complete experiments which utilized 3–10 mice per condition per experiment. Data are reported as mean ± SEM.

Figure 2

Flow cytometric analysis of LFA-1^+/+ and ^−/− neutrophils in the circulation and after extravasation into the peritoneum. Expression of (A) Mac-1 detected with mAb M1/70 biotin and (B) α4 integrin detected with mAb 9C10-biotin. Geometric means of the major peak are shown in the top right hand corner. Data are representative of five experiments with blood and lavage cells pooled from five and two mice, respectively.

Figure 2

Flow cytometric analysis of LFA-1^+/+ and ^−/− neutrophils in the circulation and after extravasation into the peritoneum. Expression of (A) Mac-1 detected with mAb M1/70 biotin and (B) α4 integrin detected with mAb 9C10-biotin. Geometric means of the major peak are shown in the top right hand corner. Data are representative of five experiments with blood and lavage cells pooled from five and two mice, respectively.

Figure 3

The effect of LFA-1 deficiency on neutrophil rolling, adhesion, and transmigration as detected by intravital microscopy. A comparison in LFA-1^+/+ and ^−/− mice of leukocyte (A) velocity (V_WBC); (B) integrated cell flux (i.e., cell flux per 10⁶ leukocytes per milliliter of blood). Inset, basic cell flux (cells per min); (C) cell adhesion; (D) emigrated leukocytes per field (“field” represents cells within 50-μm distance from a 100-μm segment of vessel). The neutrophils from LFA-1^−/− mice have a higher velocity than those from LFA-1^+/+ mice indicating a role for LFA-1 in neutrophil rolling. Because LFA-1 does not alter the cell flux (frequency of contact with endothelium), it must be decreasing the velocity by transiently stabilizing the rolling cells. A lack of LFA-1 is also reflected in lower adhesion per 100-μm length of mesenteric vessel and decreased emigration from the vasculature by the LFA-1^−/− as compared with LFA-1^+/+ neutrophils. Data are reported as mean ± SEM.

Figure 3

The effect of LFA-1 deficiency on neutrophil rolling, adhesion, and transmigration as detected by intravital microscopy. A comparison in LFA-1^+/+ and ^−/− mice of leukocyte (A) velocity (V_WBC); (B) integrated cell flux (i.e., cell flux per 10⁶ leukocytes per milliliter of blood). Inset, basic cell flux (cells per min); (C) cell adhesion; (D) emigrated leukocytes per field (“field” represents cells within 50-μm distance from a 100-μm segment of vessel). The neutrophils from LFA-1^−/− mice have a higher velocity than those from LFA-1^+/+ mice indicating a role for LFA-1 in neutrophil rolling. Because LFA-1 does not alter the cell flux (frequency of contact with endothelium), it must be decreasing the velocity by transiently stabilizing the rolling cells. A lack of LFA-1 is also reflected in lower adhesion per 100-μm length of mesenteric vessel and decreased emigration from the vasculature by the LFA-1^−/− as compared with LFA-1^+/+ neutrophils. Data are reported as mean ± SEM.

Figure 3

The effect of LFA-1 deficiency on neutrophil rolling, adhesion, and transmigration as detected by intravital microscopy. A comparison in LFA-1^+/+ and ^−/− mice of leukocyte (A) velocity (V_WBC); (B) integrated cell flux (i.e., cell flux per 10⁶ leukocytes per milliliter of blood). Inset, basic cell flux (cells per min); (C) cell adhesion; (D) emigrated leukocytes per field (“field” represents cells within 50-μm distance from a 100-μm segment of vessel). The neutrophils from LFA-1^−/− mice have a higher velocity than those from LFA-1^+/+ mice indicating a role for LFA-1 in neutrophil rolling. Because LFA-1 does not alter the cell flux (frequency of contact with endothelium), it must be decreasing the velocity by transiently stabilizing the rolling cells. A lack of LFA-1 is also reflected in lower adhesion per 100-μm length of mesenteric vessel and decreased emigration from the vasculature by the LFA-1^−/− as compared with LFA-1^+/+ neutrophils. Data are reported as mean ± SEM.

Figure 3

The effect of LFA-1 deficiency on neutrophil rolling, adhesion, and transmigration as detected by intravital microscopy. A comparison in LFA-1^+/+ and ^−/− mice of leukocyte (A) velocity (V_WBC); (B) integrated cell flux (i.e., cell flux per 10⁶ leukocytes per milliliter of blood). Inset, basic cell flux (cells per min); (C) cell adhesion; (D) emigrated leukocytes per field (“field” represents cells within 50-μm distance from a 100-μm segment of vessel). The neutrophils from LFA-1^−/− mice have a higher velocity than those from LFA-1^+/+ mice indicating a role for LFA-1 in neutrophil rolling. Because LFA-1 does not alter the cell flux (frequency of contact with endothelium), it must be decreasing the velocity by transiently stabilizing the rolling cells. A lack of LFA-1 is also reflected in lower adhesion per 100-μm length of mesenteric vessel and decreased emigration from the vasculature by the LFA-1^−/− as compared with LFA-1^+/+ neutrophils. Data are reported as mean ± SEM.

Figure 4

The effect of mAbs specific for LFA-1, Mac-1, and α4 integrin on neutrophil rolling, adhesion, and migration in LFA-1^+/+ and ^−/− mice. For LFA-1^+/+ mice, anti–LFA-1 mAb significantly affected all three stages of rolling, adhesion, and emigration and anti-α4 mAb had significant impact on cell rolling and adhesion. Anti–Mac-1 mAb significantly affected the emigration step in LFA-1^+/+ but not LFA-1^−/− mice. In LFA-1^−/− mice, only the α4 mAb significantly affects all three steps of rolling velocity, adhesion, and emigration. Data are reported as mean ± SEM.