β 2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function

Abstract

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of β2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of β2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two β2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting β2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

Keywords: CD11b/CD18 integrin; neutrophil function; neutrophils; therapeutic targets; β2 integrin signaling.

Figure 1

Inside-out signaling recruits Talin-1 and Kindlin-3 to β2 integrin. Upon stimulation of neutrophils with chemokines, both Talin-1 and Kindlin-3 become rapidly recruited to activate β2 integrins. This can be divided into two pathways for Talin-1 and Kindlin-3. For Talin-1 recruitment (A), GPCRs engage with their ligands which causes the heterotrimeric G-proteins to split into G_α and G_βγ. This initiates the activation of PLC by G_{α i}, and G_βγ. Signaling through the second messengers’ calcium which is released from the endoplasmic reticulum by IP₃ and diacylglycerol (DAG) is a critical element in many biological systems. Integration of calcium and DAG signals has been suggested to occur primarily through protein kinase C (PKC) family members, which bind both calcium and DAG. An alternative pathway involves CalDAG-GEF1, which has structural features (calcium-binding EF hands and DAG-binding C1 domains) and functions in calcium and DAG signal integration. This GEF activates RAP1, which then results into activation of RIAM and binding of RIAM to Talin-1. Together with catalyzation of PIP into PI(4,5)P₂ by PIP5K_γ90 through the PKC-PLD-Arf6 axis, these events recruit Talin-1 to β2 integrin, thus extending the integrin from the bent conformation. G_βγ is also involved in Kindlin-3 recruitment (B). In addition to activating PLC, G_βγ also activates PI3K, which generates PI(3,4,5)P₃. This allows recruitment of Kindlin-3 to β2 integrin. Binding of Kindlin-3 after Talin-1 binding activates extended β2 integrins. Note that the Kindlin-3 recruitment cascade might not be complete.

Figure 2

Outside-in signaling involves different protein complexes in response to neutrophil effector function. Once β2 integrin is activated on neutrophils, outside-in signaling is facilitated in order to induce cell effector function. Neutrophils are endowed with multiple mechanism, all achieving target elimination. In all cases, integrin outside-in signaling involves stable interaction with Talin-1 and Kindlin-3, which recruits binding of the actin cytoskeleton and induction of signaling via Sarcoma family kinases (SFKs). During spreading (A), SFKs phosphorylate and recruit SYK to the cytoplasmic tail of the β2 integrin, while Phosphatidylinositol 4-phosphate 5-kinase (PIP5KIC) induces activation of RhoA GTPase. Mitogen-activated binding protein 1 (mAbp1) is involved in stabilization of actin cytoskeleton, while Calpain-1 could play a role to negatively regulate its function. During migration (B), phosphorylated PYK2 is recruited and the Arp2/3 complex is stabilized via SKAP2 and Rac2 GTPase. Filamin A and ARPC1B are important for migration. We do not show WASP protein because deficiency results in impaired neutrophil migration in murine models only. Particularly during phagocytosis (C), phosphorylation of tyrosine protein kinase SYK induces binding of phosphorylated Paxillin with subsequent activation of Vinculin and activation of Phosphoinositide 3-kinase (PI3Kβ) and mAbp1. SCAR1/2 protein (also known as WAVE1/2) recruits Arp2/3 complex to stabilize actin cytoskeleton, while SKAP2 (or SKAP55R) functions as adaptor protein. During ROS production (D), degranulation is facilitated through increase of calcium influx. Phosphorylation of SYK initiates VAV phosphorylation with ADAP1 and PLCγ2 complex formation, promoting oxidative burst. VAV induces Rac1 GTPase activity, which facilitates NAPDH oxidase formation. In this context, Calpain1 could additionally inhibit Talin-1 or Focal adhesion kinase (FAK) function.