Vav/Phospholipase Cgamma2-mediated control of a neutrophil-dependent murine model of rheumatoid arthritis

Abstract

Objective: Accumulating evidence indicates an important role of neutrophils in the development of rheumatoid arthritis (RA). Recruitment of neutrophils to the joint space and release of proteolytic enzymes can exacerbate tissue damage and the inflammatory response related to RA. Engagement of beta2 integrin and subsequent activation of downstream signaling have been shown to be fundamental for activation of neutrophil effector functions. The aim of this study was to test the hypothesis that Vav and phospholipase Cgamma2 (PLCgamma2), two molecules involved in integrin signaling, are required for arthritis generation and neutrophil activation in a mouse model of arthritis.

Methods: Arthritis was induced in wild-type (WT), Vav(null), and PLCgamma2(-/-) mice using the K/BxN serum-transfer model. Neutrophil function was assessed by analyses of adhesion, spreading, and degranulation on integrin-dependent substrates. Regulation of integrin signaling was determined by analyzing the phosphorylation of Pyk-2, Src, and ERK.

Results: Vav(null) and PLCgamma2(-/-) mice were protected from inflammation and bone erosion in the K/BxN serum-transfer model of arthritis. Mechanistically, Vav and PLCgamma2 control neutrophils mediated spreading and degranulation on integrin-dependent substrates. Consequently, the Vav/PLCgamma2 axis, acting downstream of the integrin receptor, modulated the activation of Pyk-2, Src, and ERK.

Conclusion: Our findings show that Vav cooperates with PLCgamma2 in modulating neutrophil activation downstream of the integrin receptor. This study identifies a Vav/PLCgamma2-dependent signaling pathway as a possible therapeutic target for the treatment of inflammation and bone disruption in arthritis.

Figure 1

Protection of PLCγ2^−/− mice from inflammatory arthritis. A, Hind paw thickness measured daily in wild-type (WT) and PLCγ2^−/− mice injected intraperitoneally with serum from K/BxN mice. Increase in paw swelling was expressed as the fold induction from baseline. Values are the mean and SD of 4 mice per group. Results from 1 of 3 representative experiments are shown. B, Histologic features of hind paws obtained on day 14 from WT and PLCγ2^−/− mice treated as in A. Sections were stained with hematoxylin and eosin (H&E) to detect inflammatory infiltrates (*) or with tartrate-resistant acid phosphatase (TRAP) to detect osteoclasts (arrowheads). Arrows indicate bone erosion (original magnification × 10). C, Levels of expression of mRNA for the inflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor α (TNFα) in tissue extracts from paws obtained on day 7 from WT and PLCγ2^−/− mice injected with arthritogenic serum. Data were normalized for GAPDH expression. Values are the mean and SD of at least 4 mice per group. D, Levels of expression of mRNA for the neutrophil marker Gr-1 in tissue extracts from paws obtained on day 7 from WT and PLCγ2^−/− mice treated as in C. Values are the mean and SD of 4 mice per group.

Figure 2

Protection of Vav^null mice from inflammatory arthritis. A, Hind paw thickness measured daily in WT and Vav^null mice injected intraperitoneally with serum from K/BxN mice. Increase in paw swelling was expressed as the fold induction from baseline. Values are the mean and SD of 4 mice per group. Results from 1 of 3 representative experiments are shown. B, Histologic features of hind paws obtained on day 14 from WT and Vav^null mice treated as in A. Sections were stained with hematoxylin and eosin (H&E) to detect inflammatory infiltrates (*) or with tartrate-resistant acid phosphatase (TRAP) to detect osteoclasts (arrowheads). Arrows indicate bone erosion (original magnification × 10). C, Levels of expression of mRNA for the inflammatory cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor α (TNFα) in tissue extracts from paws obtained on day 7 from WT and Vav^null mice injected with arthritogenic serum. Data were normalized for GAPDH expression. Values are the mean and SD of at least 4 mice per group. D, Levels of expression of mRNA for the neutrophil marker Gr-1 in tissue extracts from paws obtained on day 7 from WT and Vav^null mice treated as in C. Values are the mean and SD of 4 mice per group.

Figure 3

Failure of Vav and PLCγ2 to control neutrophil chemotaxis. A and B, In vitro Transwell migration of neutrophils from wild-type (WT), Vav^null, and PLCγ2^−/− mice in response to the indicated concentrations of C5a. Values are the mean and SD. Results are representative of 3 independent experiments. C, In vivo migration of neutrophils from WT and PLCγ2^−/− mice to the peritoneum following a single intraperitoneal injection of 4% thioglycollate. Values are the mean and SD number of peritoneal exudate cells. Results are representative of 3 independent experiments.

Figure 4

Requirement of Vav and PLCγ2 for cell spreading in response to integrin stimuli. A, Adhesion of neutrophils from wild-type (WT), Vav^null, and PLCγ2^−/− mice to pRGD or in response to phorbol myristate acetate (PMA; positive control). Values are the mean and SD. * = P < 0.05 versus WT cells. B, Phalloidin immunostaining of cells treated as in A to detect actin organization (original magnification × 20). C, Capacity of neutrophils to spread on pRGD or in response to PMA. Spreading was defined as cells having lamellipodia, as indicated by phalloidin staining. Values are the mean and SD percentage of cells that had spread versus the total cell number. * = P < 0.05 versus WT cells.

Figure 5

Failure of neutrophils from Vav^null and PLCγ2^−/− mice to undergo adhesion-mediated degranulation. Degranulation of neutrophils from wild-type (WT), Vav^null, and PLCγ2^−/− mice in response to A, pRGD and phorbol myristate acetate (PMA), B, C5a and fibrinogen (Fgn), and C, tumor necrosis factor α (TNFα) and fibrinogen was determined by Western blotting (WB), according to the release of lactoferrin into the supernatant (Sup). The lactoferrin content in the cell lysate was also determined. Results are representative of 5 independent experiments. Untx = untreated.

Figure 6

Requirement of Vav and PLCγ2 for Pyk-2, Src, and ERK activation of neutrophils. Activation of the phosphorylated forms of Pyk-2, Src, and ERK in neutrophils from wild-type (WT), Vav^null, and PLCγ2^−/− mice was determined by Western blotting of cells plated on pRGD for 5, 10, or 15 minutes. β-actin served as a loading control. Results are representative of 3 independent experiments.