Title

Vav1 Is Essential for Mechanotactic Crawling and Migration of Neutrophils out of the Inflamed Microvasculature

Document Type

Article

Abstract

Mac-1-dependent crawling is a new step in the leukocyte recruitment cascade that follows LFA-1-dependent adhesion and precedes emigration. Neutrophil adhesion via LFA-1 has been shown to induce cytoskeletal reorganization through Vav1-dependent signaling, and the current study investigates the role of Vav1 in the leukocyte recruitment process in vivo with particular attention to the events immediately downstream of LFA-1-dependent adhesion. Intravital and spinning-disk-confocal microscopy was used to investigate intravascular crawling in relation to endothelial junctions in vivo in wild-type and Vav1−/− mice. Adherent wild-type neutrophils almost immediately began crawling perpendicular to blood flow via Mac-1 until they reached an endothelial junction where they often changed direction. This pattern of perpendicular, mechanotactic crawling was recapitulated in vitro when shear was applied. In sharp contrast, the movement of Vav1−/− neutrophils was always in the direction of flow and appeared more passive as if the cells were dragged in the direction of flow in vivo and in vitro. More than 80% of Vav1−/− neutrophils moved independent of Mac-1 and could be detached with LFA-1 Abs. An inability to release the uropod was frequently noted for Vav1−/− neutrophils, leading to greatly elongated tails. The Vav1−/− neutrophils failed to stop or follow junctions and ultimately detached, leading to fewer emigrated neutrophils. The Vav1−/− phenotype resulted in fewer neutrophils recruited in a relevant model of infectious peritonitis. Clearly, Vav1 is critical for the complex interplay between LFA-1 and Mac-1 that underlies the programmed intravascular crawling of neutrophils.

Recruitment of circulating leukocytes to the site of inflammation occurs via complex interactions with endothelium; leukocytes tether to, roll along, and firmly adhere to the endothelium before transmigrating out of the vasculature (1, 2). In the case of neutrophils, rolling is dependent on selectins, adhesion occurs mainly through β2 integrins, and emigration is mediated by integrins as well as PECAM-1, CD99, and junction adhesion molecules (3, 4, 5, 6, 7). Schenkel et al. (8) recently documented another step in the leukocyte recruitment cascade, namely crawling to junctions before transendothelial migration. Using an in vitro system to visualize monocytes, these investigators reported that cells firmly adhered, flattened, and then rapidly sent out pseudopods and crawled via integrins to junctions where they subsequently transmigrated. The crawling appeared to be random but was ultimately necessary for emigration. More recently, a number of laboratories reported that intraluminal crawling also occurred in vivo (9, 10, 11), and this allowed cells to reach optimal emigration sites at endothelial junctions (10). The crawling of neutrophils did not appear to be biased in the direction of flow, suggesting this was not simply a dragging motion due to hydrodynamic force displacement. However, whether the neutrophil crawling was truly a random process or followed an inherent pattern remained unclear, as was the question of whether the crawling was chemotactic, chemokinetic, haptotactic (following chemokine attached to extra cellular matrix), or mechanotactic (driven by mechanical forces).

Neutrophil adhesion and crawling are mediated by two separate molecular mechanisms. Firm adhesion and stabilization (spreading) occurred via αLβ2 integrin (LFA-1), followed immediately by crawling via αMβ2 integrin (Mac-1). Inhibition of LFA-1 prevented adhesion while inhibition of Mac-1 had no effect on adhesion but prevented all subsequent crawling (10). These distinct, molecular steps for LFA-1-induced adhesion followed by Mac-1-induced crawling in vivo would require exquisite communication (e.g., outside in signaling from LFA-1) for these molecules to function in a sequential, coordinated fashion under flow conditions. Moreover, these in vivo observations bestow added physiologic importance to various in vitro studies that demonstrated that cross linking of LFA-1 leads to outside in signaling and cytoskeletal rearrangements (12, 13) as well as Mac-1 activation (14) presumably leading to crawling. However, intracellular signaling molecules that might be important downstream of adhesion to impact on crawling and resultant emigration have not been identified.

Vav1, a guanine exchange factor for the Rho family GTPases Rac and Cdc42, is a major regulator of the organization of the actin cytoskeleton during leukocyte polarization and migration (15). Importantly, Vav1 regulates activation downstream of LFA-1 in all leukocytes tested, including neutrophils, lymphocytes, and NK cells (13, 16, 17, 18). Absence of Vav1 under static conditions in vitro leads to impaired neutrophil spreading, membrane ruffling, cytoskeletal rearrangement, and polarization. However, the role for Vav1 in crawling becomes equivocal under static conditions in vitro. One group has reported no role for Vav1 in crawling on protein coated coverslips (19), another group observed reduced crawling speed for Vav1−/− cells on plastic (20), whereas a third group reported impaired crawling to some but not other chemoattractants (21). To date, no one has examined the role of Vav1 for crawling under the very dynamic shear-dependent in vivo leukocyte recruitment paradigm. In fact, to our knowledge, only one group has visualized leukocyte behavior in vivo in Vav1-deficient mice by adding a brief chemotactic stimulus and examined neutrophil rolling and adhesion for the subsequent 10 min in real time (19). The data revealed no obvious impairment in rolling or adhesion in this very acute setting in Vav1−/− neutrophils; however, events downstream of adhesion were not examined.

Using intravital light and multichannel fluorescence spinning disk confocal microscopy in real time and time-lapse, we systematically examined the transition from adhesion to crawling to emigration in wild-type (WT)4 and Vav1−/− mice. A very striking and consistent Mac-1-dependent crawling behavior was noted in WT mice, which was often perpendicular to blood flow but sometimes changed when an endothelial junction was encountered. In vitro, similar perpendicular crawling was observed in the presence of shear and absence of chemokine gradients on protein-coated coverslips, suggesting mechanotaxis rather than chemotaxis for the inherent perpendicular crawling behavior. In striking contrast, Vav1−/− neutrophils were unable to crawl perpendicular to the direction of blood flow in vitro and in vivo under physiological shear rates. In fact, adherent Vav1−/− neutrophils appeared to be stretched and dragged in the direction of blood flow. This movement in Vav1−/− mice was Mac-1 independent. Vav1−/− cells also failed to realign and follow any junctions that were not aligned in the direction of flow. This resulted in a greater interval of time for adhesion and crawling in Vav1−/− mice, leading to more detachment and reduced emigration. Finally, these neutrophil recruitment defects translated into significant impairment in recruitment of neutrophils in an infectious disease relevant model of peritonitis.

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