Factor XII and uPAR upregulate neutrophil functions to influence wound healing
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier
View: Text | PDF
Research Article Cell biology Inflammation

Factor XII and uPAR upregulate neutrophil functions to influence wound healing

Abstract

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Authors

Evi X. Stavrou, Chao Fang, Kara L. Bane, Andy T. Long, Clément Naudin, Erdem Kucukal, Agharnan Gandhi, Adina Brett-Morris, Michele M. Mumaw, Sudeh Izadmehr, Alona Merkulova, Cindy C. Reynolds, Omar Alhalabi, Lalitha Nayak, Wen-Mei Yu, Cheng-Kui Qu, Howard J. Meyerson, George R. Dubyak, Umut A. Gurkan, Marvin T. Nieman, Anirban Sen Gupta, Thomas Renné, Alvin H. Schmaier

×

Figure 9

FXII signaling in neutrophils regulates αMβ2 surface expression, intracellular Ca2+ mobilization, and NET formation.

Options: View larger image (or click on image) Download as PowerPoint
FXII signaling in neutrophils regulates αMβ2 surface expression, intrace...
(A) Surface expression of αMβ2 integrin on UT (grey curve) and FXII/Zn2+–stimulated (pink curve) neutrophils. Representative flow diagram of n = 7 individual experiments. (B) Quantitation of αMβ2 antibody binding (mean fluorescent intensity [MFI]). Mean ± SEM, n = 7 experiments. *P < 0.03 UT vs. FXII/Zn2+, 1-way ANOVA. (C) WT neutrophils were loaded with 1 μM Fluro-4-AM Ca2+ dye for 45 minutes and then treated with or without 5 mM ATP, 3 μM ionomycin, 10 μM fMLP, FXII (62 nM or 100 nM)/10 μM Zn2+, or Zn2+ alone. Intracellular Ca2+ mobilization was measured at 30-second intervals for 30 minutes. (D) Intracellular Ca2+ concentration with various agonists. Mean ± SEM, n = 4, each run in triplicate. *P < 0.0001 vs. UT cells, 1-way ANOVA. (E) WT neutrophils were stimulated with fMLP, 100 nM PMA, or FXII/Zn2+ for 120 minutes, and immunoblot analysis was performed for H3-C. Representative blot of n = 4. (F) H3-C fold increase compared with UT neutrophils. Mean ± SEM, n = 4. *P < 0.007 UT vs. FXII/Zn2+, 1-way ANOVA. (G) WT neutrophils were stimulated with 62 nM FXII/10 μM Zn2+ for the indicated times. Immunoblot analysis was performed for H3-C. Representative blot of n = 4. (H) H3-C fold increase compared with UT neutrophils. Mean ± SEM, n = 4. *P = 0.007 at 30 minutes; **P < 0.002 at 60 minutes; ***P = 0.03 at 120 minutes vs. UT, 1-way ANOVA. (I) NETotic index rate of WT neutrophils activated with fMLP, 4 μM A23187, 1 μM PMA, FXII/Zn2+, or Zn2+ alone. Where indicated, neutrophils were preincubated with 5 μM Akti XII or 300 μM LRG20 for 30 minutes before stimulation with FXII/ Zn2+. Curves represent mean ± SEM except A23187 (shown as mean). n = 5–9 each run in triplicate. *P < 0.0001 FXII/ Zn2+ vs. media at 150–400 minutes, 2-way ANOVA.