Vav/Phospholipase Cγ2–mediated control of a neutrophil‐dependent murine model of rheumatoid arthritis

Rheumatoid arthritis (RA) is a debilitating chronic autoimmune disease characterized by progressive inflammation that affects the synovial membranes of the joints. Accumulating evidence suggests that the innate branch of the immune system is fundamental in the induction of arthritis (1). The K/BxN serum–transfer model of inflammatory arthritis bears clinical and histopathologic similarities to human RA, such as the recruitment of leukocytes to the joint space, pannus formation, and bone and cartilage damage (2). Transfer of serum from K/BxN mice containing autoantibodies directed toward glucose-6-phosphate isomerase into a recipient mouse leads to the development of an inflammatory arthritis that is strongly dependent on neutrophils (3). In particular, neutrophils are recruited to the joint space within 1–2 days from the initial injection of arthritic serum. Within 1 week, synovial inflammation, pannus formation, and erosion of bone and cartilage are observed (4). A crucial mechanism for the induction of inflammation includes the release of neutrophil granules that contain proteolytic enzymes, such as elastase, matrix metalloproteinases, and collagenase, that can exacerbate the tissue damage and amplify the neutrophil response (5). Regulation of neutrophil activity is largely dependent on integrin-mediated adhesive interactions (6,7). Integrins are transmembrane heterodimeric glycoproteins consisting of an α/β heterodimer that mediates cell–cell and cell–extracellular matrix interactions (8) and transmits intracellular signals, leading to gene expression and cytoskeleton rearrangement (9,10). In the immune system, integrins play essential roles in leukocyte trafficking and function (11). These include immune cell attachment to endothelial and antigen-presenting cells, cytotoxicity, and extravasation into tissues, all of which are essential functions for activation of the inflammatory response (12). In particular, β2 integrins are implicated in the regulation of neutrophil effector functions, such as bacterial clearance, oxidative burst, and degranulation (7,13). The Vav family of guanine nucleotide exchange factors mediates signaling downstream of integrin receptors by promoting the activation of Rho family GTPases, thereby modulating the reorganization of the cytoskeleton in response to cell adhesion (14–17). Three Vav proteins exist: Vav1 is expressed primarily in hematopoietic cells, whereas Vav2 and Vav3 are more broadly expressed (18). In neutrophils, Vav1 and Vav3 regulate β2 integrin–mediated adhesion and signaling through activation of Pyk-2, Src, and ERK (15). In a recent study, we also showed that Vav proteins control the phosphorylation of phospholipase Cγ2 (PLCγ2) downstream of integrin signaling, thereby modulating the induction of neutrophil oxidative burst by a variety of adhesion-dependent stimuli (19). PLCγ has been implicated in the activation of integrin receptor signaling in various cell types (20,21). Two isoforms of PLCγ are known: PLCγ1, which is widely expressed, and PLCγ2, which is more specifically expressed in the hematopoietic lineage (22). While PLCγ1 participates in T cell receptor signals, where it modulates calcium fluxes, PLCγ2 has been linked to integrin function in platelets, where its phosphorylation downstream of the αIIbβ3 integrin is necessary for platelet spreading (23). We have also observed that PLCγ2 mediates αvβ3 integrin signaling in osteoclasts, which is required for efficient bone resorption (24). In the present study, we show that Vav1/2/3 triple-knockout (Vavnull) mice and PLCγ2−/− mice are protected from inflammatory arthritis in the K/BxN serum–transfer model. Using neutrophils from Vavnull and PLCγ2−/− mice, we demonstrated that the Vav/PLCγ2 pathway controls integrin-dependent neutrophil functions, such as spreading and degranulation. We also show that Vav and PLCγ2 are required for β2 integrin–mediated phosphorylation of Pyk-2, Src, and ERK. Our data suggest that targeting the Vav/PLCγ2 signaling axis might be of clinical relevance for amelioration of arthritis.