DOCK2 and DOCK5 act additively in neutrophils to regulate chemotaxis, superoxide production, and extracellular trap formation

Abstract

Neutrophils are highly motile leukocytes that play important roles in the innate immune response to invading pathogens. Neutrophils rapidly migrate to the site of infections and kill pathogens by producing reactive oxygen species (ROS). Neutrophil chemotaxis and ROS production require activation of Rac small GTPase. DOCK2, an atypical guanine nucleotide exchange factor (GEF), is one of the major regulators of Rac in neutrophils. However, because DOCK2 deficiency does not completely abolish fMLF-induced Rac activation, other Rac GEFs may also participate in this process. In this study, we show that DOCK5 acts with DOCK2 in neutrophils to regulate multiple cellular functions. We found that fMLF- and PMA-induced Rac activation were almost completely lost in mouse neutrophils lacking both DOCK2 and DOCK5. Although β2 integrin-mediated adhesion occurred normally even in the absence of DOCK2 and DOCK5, mouse neutrophils lacking DOCK2 and DOCK5 exhibited a severe defect in chemotaxis and ROS production. Similar results were obtained when human neutrophils were treated with CPYPP, a small-molecule inhibitor of these DOCK GEFs. Additionally, we found that DOCK2 and DOCK5 regulate formation of neutrophil extracellular traps (NETs). Because NETs are involved in vascular inflammation and autoimmune responses, DOCK2 and DOCK5 would be a therapeutic target for controlling NET-mediated inflammatory disorders.

FIGURE 1

DOCK2 and DOCK5 act additively in regulation of GPCR-mediated Rac activation. (A) Expression of DOCK-A family proteins in BM neutrophils, splenocytes, and mouse embryonic fibroblasts (MEFs). Cell lysates were subjected to immunoblotting using anti-DOCK1, -DOCK2, and -DOCK5 Abs. Data are representative of two independent experiments. (B) Flow cytometric analysis for the expression of Gr-1 and CD11b on BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice. Numbers indicate the percentages of Gr-1⁺CD11b⁺ cells in purified BM neutrophils. Data are representative of more than three independent experiments. (C and D) Activation of Rac1 and Rac2 in BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice stimulated with fMLF (10 μM) for the indicated times. Cell lysates were subjected to pull-down assays using GST-fusion Rac-binding domain of PAK1 before immunoblotting with anti-Rac1 and -Rac2 Abs. Results were quantified by densitometry and are expressed as the ratio of the GTP-bound form to total protein after normalization of the 15 s value of WT neutrophils to an arbitrary value of 1. Data are indicated as means ± SEM of three separate experiments. *p < 0.05, **p < 0.01. (E) Expression of DOCK2, DOCK5, P-Rex1, and Vav proteins in BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice. Cell lysates were subjected to immunoblotting using anti-DOCK2, -DOCK5, –P-Rex1, and -Vav Abs. Data are representative of two independent experiments. (F and G) Phosphorylations of ERK, Akt, and p38 in BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice stimulated with fMLF (10 μM) for the indicated times. Cell lysates were subjected to immunoblotting using phosphorylation-specific Abs against ERK, Akt, and p38. Results were quantified by densitometry and are expressed as the ratio of phosphorylated form to total protein after normalization of the WT value (2 min value for ERK and Akt, 5 min value for p38) to an arbitrary value of 1. Data are indicated as means ± SEM of three separate experiments. *p < 0.05, **p < 0.01.

FIGURE 2

DOCK2 and DOCK5 additively regulate neutrophil chemotaxis and ROS production. (A) Mouse BM neutrophils chemotaxing under fMLF gradient (0–10 μM) were analyzed using an EZ-TAXIScan chamber. Data were collected at 30-s intervals for 20 min. Data are representative of three independent experiments (n = 51–100/group). Each box exhibits the median (central line within each box), the 25th and 75th percentile values (box end), and the 10th and 90th percentile values (error bar). **p < 0.01. (B) BM neutrophils chemotaxing under CXCL2 gradient (0–1 μg/ml) were similarly analyzed as in (A). (C) BM neutrophils chemotaxing under the fMLF gradient were stained with phalloidin, and the percentages of neutrophils with polarized F-actin localization were compared. DIC, differential interference contrast. Scale bar, 10 μm. Data are indicated as means ± SEM of five separate experiments. **p < 0.01. Cells are judged to be positive when F-actin staining is confined to less than one third of the circumference. (D and E) ROS production was compared among WT, DOCK2^−/−, DOCK5^−/−, and DKO neutrophils stimulated with fMLF (10 μM). In (D), the right panel indicates the magnified view of the graph to show the difference between DOCK2^−/− and DKO neutrophils. In (E), results are expressed as the ratio after normalization of the WT value to an arbitrary value of 1. Data are indicated as means ± SD of triplicate samples. **p < 0.01.

FIGURE 3

Treatment of human neutrophils with CPYPP inhibits Rac2 activation, chemotaxis, and ROS production. (A and B) Following treatment with CPYPP (100 μM) or vehicle (DMSO) alone for 1 h, human peripheral blood neutrophils were stimulated with fMLF (10 μM) for the indicated times. Cell lysates were subjected to pull-down assays using the GST-fusion Rac-binding domain of PAK1 before immunoblotting using anti-Rac2 Ab. Results were quantified by densitometry and are expressed as the ratio after normalization of the 5 s value of vehicle-treated sample to an arbitrary value of 1. Data are indicated as means ± SEM of three separate experiments. **p < 0.01. (C and D) Human peripheral blood neutrophils were treated with CPYPP at the indicated concentrations and their chemotactic responses under fMLF gradient (0–10 μM) were analyzed using an EZ-TAXIScan chamber. Data were collected at 30-s intervals for 20 min. Data are representative of three separate experiments (n = 40/group). Each box exhibits the median (central line within each box), the 25th and 75th percentile values (box end), and the 10th and 90th percentile values (error bar). **p < 0.01. (E) ROS production in response to fMLF (10 μM) stimulation was compared between human neutrophils treated with CPYPP (100 μM) and vehicle alone. Results are expressed as the ratio after normalization of the value of vehicle-treated sample to an arbitrary value of 1. Data are indicated as means ± SD of triplicate samples. **p < 0.01.

FIGURE 4

PMA-induced Rac activation and ROS production are also defective in DKO neutrophils. (A and B) Activation of Rac1 and Rac2 in BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice stimulated with PMA (100 nM) for the indicated times. Cell lysates were subjected to pull-down assays using GST-fusion Rac-binding domain of PAK1 before immunoblotting with anti-Rac1 and -Rac2 Abs. Results were quantified by densitometry and are expressed as the ratio of GTP-bound form to total protein after normalization of the 2 min value of WT neutrophils to an arbitrary value of 1. Data are indicated as means ±SEM of three separate experiments. *p <0.05, **p <0.01. (C and D) ROS production was compared among WT, DOCK2^−/−, DOCK5^−/−, and DKO neutrophils stimulated with PMA (324.24 nM). In (C), the right panel indicates the magnified view of the graph to show the difference between DOCK2^−/− and DKO neutrophils. In (D), results are expressed as the ratio after normalization of the WT value to an arbitrary value of 1. Data are indicated as means ±SD of triplicate samples. **p < 0.01. (E) ROS production by WT, DOCK2^−/−, DOCK5^−/−, and DKO neutrophils stimulated with PMA (324.24 nM) was measured by means of a cytochrome c reduction assay. Data are indicated as means ± SD of triplicate samples. *p < 0.05, **p < 0.01. (F and G) Phosphorylations of ERK and MEK in BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice stimulated with PMA (100 nM) for the indicated times. Cell lysates were subjected to immunoblotting using phosphorylation-specific Abs against ERK and MEK. Results were quantified by densitometry and are expressed as the ratio of phosphorylated form to total protein after normalization of the 5 min value of WT neutrophils to an arbitrary value of 1. Data are indicated as means ± SEM of three separate experiments.

FIGURE 5

DKO neutrophils exhibit a severe defect in NET formation. (A) Following stimulation with PMA (20 nM) for 18 h at 37°C, WT, DOCK2^−/−, DOCK5^−/−, and DKO neutrophils were stained with Sytox Green and Sytox Orange for visualization of NETs (original magnification ×200). (B) NET formation was quantitatively compared among PMA (20 nM)-stimulated neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice. Data are indicated as means ± SEM of five separate experiments. **p < 0.01.

FIGURE 6

Neutrophils lacking DOCK2 and DOCK5 are capable of normal adhesion. BM neutrophils from WT, DOCK2^−/−, DOCK5^−/−, and DKO mice were incubated on the ICAM-1 or C3bi-coated plates for 30 min at 37°C in the presence or absence of fMLF (10 μM) or PMA (162.12 nM). After being washed twice with HBSS containing 20 mM HEPES (pH 7.4) and 0.1% BSA, the percentage of adherent cells was compared among WT, DOCK2^−/−, DOCK5^−/−, and DKO neutrophils. Data are indicated as means ± SEM of three independent experiments.