Down-regulation of Rac Activity during β2 Integrin-mediated Adhesion of Human Neutrophils

Abstract

In human neutrophils, ␤ 2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the ␤ 2 integrin-induced down-regulation of Rac activities. In contrast, ␤ 2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the ␤ 2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTPbound Rac from the detergent-soluble fraction. The ␤ 2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47 phox and p67 phox , were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by ␤ 2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a ␤ 2 integrin-induced targeting of the Rac GTPases as well as p47 phox and p67 phox to the plasma membrane.

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

“…5B). This fits with observations by Dib et al [27], who showed that engagement of b 2 integrins induced an increase of GTPbound Cdc42. Moreover, inhibition of complement by sCR1 reduced activation of neutrophil Cdc42 in the immune system (Fig.…”

“…5A). However, Dib et al [27] demonstrated that although engagement of b 2 integrins resulted in a decrease of the GTP-bound form of Rac2, it caused translocation of Rac2 to the plasma membrane, where it can complex with p47 phox and p67 phox to form the active NADPH oxidase [28]. Therefore, an increase of the total amount of GTP-bound Rac2 may not be necessary for induction of respiratory burst.…”

“…Serum was used immediately or stored at -80 C until use. Neutrophils were isolated from fresh heparinized blood as described [27]. Neutrophils were resuspended in RPMI 1640 with glutamax.…”

“…The neutrophils were then lysed as described [27]. A GST fusion protein expressing the Rac/Cdc42-binding domain of PAK1B [43] was added and the samples were incubated on ice for 5 min and then centrifuged (14,000Âg, 10 min, 4 C).…”

Common and Diverging Integrin Signals Downstream of Adhesion and Mechanical Stimuli and Their Interplay with Reactive Oxygen Species

“…Mitochondria-derived ROS (52,66) as well as ROS from NOXes and 5-lipoxygenase (5-LOX) produced in response to integrin ligand binding (67,68) were reported to affect cell attachment, spreading, and associated cytoskeletal changes. There are also indications that FAK, important for survival and migration, is regulated by ROS in response to integrin-mediated adhesion e.g.…”