Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade

Abstract

The prevailing view is that the beta2-integrins Mac-1 (alphaMbeta2, CD11b/CD18) and LFA-1 (alphaLbeta2, CD11a/CD18) serve similar biological functions, namely adhesion, in the leukocyte recruitment cascade. Using real-time and time-lapse intravital video-microscopy and confocal microscopy within inflamed microvessels, we systematically evaluated the function of Mac-1 and LFA-1 in the recruitment paradigm. The chemokine macrophage inflammatory protein-2 induced equivalent amounts of adhesion in wild-type and Mac-1-/- mice but very little adhesion in LFA-1-/- mice. Time-lapse video-microscopy within the postcapillary venules revealed that immediately upon adhesion, there is significant intraluminal crawling of all neutrophils to distant emigration sites in wild-type mice. In dramatic contrast, very few Mac-1-/- neutrophils crawled with a 10-fold decrease in displacement and a 95% reduction in velocity. Therefore, Mac-1-/- neutrophils initiated transmigration closer to the initial site of adhesion, which in turn led to delayed transmigration due to movement through nonoptimal emigration sites. Interestingly, the few LFA-1-/- cells that did adhere crawled similarly to wild-type neutrophils. Intercellular adhesion molecule-1 but not intercellular adhesion molecule-2 mediated the Mac-1-dependent crawling. These in vivo results clearly delineate two fundamentally different molecular mechanisms for LFA-1 and Mac-1 in vivo, i.e., LFA-1-dependent adhesion followed by Mac-1-dependent crawling, and both steps ultimately contribute to efficient emigration out of the vasculature.

Figure 1.

In vivo leukocyte–endothelial cell interactions in inflamed venules in response to MIP-2. Rolling flux (A), rolling cell velocity (B), and adhesion (C) before and after addition of MIP-2 (0.5 nM) in the superfusate. A leukocyte was considered to be adherent if it remained stationary for more than 30 s, and total leukocyte adhesion was quantified as the number of adherent cells within a 100 μm length of venule during 5 min. All values are means of n = 6 or 7 ± SEM. ^†P < 0.05 compared with C57BL/6 mice in A and B, compared with LFA-1^−/− mice in C.

Figure 2.

In vivo adhesion and emigration in response to high-dose MIP-2. Leukocyte adhesion (A) and emigration (B) before and after addition of 5 nM MIP-2 to the superfusate. Leukocyte emigration was defined as the number of cells in the extra vascular space within a 200 × 300-μm area. All values are means of n = 5 ± SE. ^†P < 0.05 compared with C57BL/6 mice.

Figure 3.

Intravascular crawling of leukocytes. Time-lapse microscopy of inflamed postcapillary venules allows for observation of intravascular crawling in response to MIP-2 (A). Dashed arrow indicates direction of blood flow; solid arrow indicates cell of interest. Bars, 2.5 μm. (B) The percentage of the adherent cells that crawled at the vessel wall before transmigrating in C57BL/6, Mac-1, and LFA-1 mice (KO), or C57BL/6 mice treated with blocking antibodies (Ab). (C) The displacement of adherent neutrophils from where they first started to adhere to where transmigration took place. (D) The velocity of the displacement was calculated by dividing displacement by the time the cells were followed. (E) Percentage of adherent cells which crawl in response to TNF-α superfusion. All values are means ± SEM. *P < 0.05 compared with C57BL/6.

Figure 4.

Crawling requires ICAM-1, and emigration is delayed in Mac-1 mice. (A) Crawling after ICAM-1/2 blockade, and (B) total emigration time of adherent knockout (KO) or antibody (Ab)-treated neutrophils. All values are means ± SEM. *P < 0.05 compared with C57BL/6.

Figure 5.

Confocal images of transmigrating neutrophils. Endothelial junctions are stained with anti–PECAM-1 (red), and the neutrophils are stained with anti–GR-1 (green). (A) Z-projection of a single venule demonstrating the morphology of the endothelial cells and locations of adherent and transmigrating neutrophils. (B and C) Z-projections of endothelial cells overlaid with a single z-section showing the transendothelial portion of transmigrating neutrophils in C57BL/6 (B) and Mac-1^−/− (C) mice. Cells are transmigrating at junctions (arrows) and undergoing transcellular migration (arrowhead). (D) Z-sequence of a transmigrating neutrophil moving through the middle of an endothelial cell. Each image consists of a single z-section. (I) Portion of neutrophil (green) within the lumen of the blood vessel. (II) Portion of the neutrophil extending through the endothelial cell. (III) Portion of neutrophil on the basolateral side of the endothelial cell layer. (E) Quantification of the number of transjunctional and transcellular migrating neutrophils in C57BL/6 and Mac-1^−/− mice. A–C is a representative sample of a minimum of 13 venules. (E) n = 64 (C57BL/6) and 56 (Mac-1^−/−) cells from 15 (C57BL/6) and 13 (Mac-1^−/−) venules.