Factor XII and uPAR upregulate neutrophil functions to influence wound healing
Evi X. Stavrou, … , Thomas Renné, Alvin H. Schmaier
Evi X. Stavrou, … , Thomas Renné, Alvin H. Schmaier
Published January 29, 2018
Citation Information: J Clin Invest. 2018;128(3):944-959. https://doi.org/10.1172/JCI92880.
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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

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Figure 6

FXII is secreted from neutrophils and signals through uPAR to promote pAktS473 and pAktS474.

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FXII is secreted from neutrophils and signals through uPAR to promote pA...
(A) WT neutrophils (PMNs) were incubated in the absence or presence of fMLP. At indicated time points, cells and supernatant were separated and resolved on SDS-PAGE. FXII was detected with anti-FXII antibody. Representative blot of n = 3. (B) Sensorgram of FXII binding to immobilized uPAR in the presence (solid lines) or absence (dotted lines) of 10 μM Zn2+. Increasing concentrations of FXII (0 nM, 1 nM, 10 nM, 100 nM, 400 nM) were injected over a uPAR-immobilized, gelatin-coated CM5 chip. Each sensorgram line represents an average of 3 injection runs as described in Methods. Analyte injection was terminated at 120 seconds. (C–E, top panels) PMNs were treated with fMLP, FXII/Zn2+, or Zn2+ alone for 5 minutes, followed by immunoblotting. Wort or LY lanes were pretreated with Wortmannin (50 μM) and LY294002 (100 μM) for 1 hour, respectively, followed by FXII/Zn2+ treatment. (C–E, bottom panels) Percentage pAktS473 in neutrophils. (F–H, top panels) Neutrophils were treated with fMLP, FXII/Zn2+, or FXII alone. Lanes labeled Akti were pretreated with Akti XII (5 μM) for 30 minutes, followed by FXII/Zn2+ treatment. Where indicated, cells were pretreated with TPEN (10 μM) for 30 minutes before stimulation with fMLP or FXII/Zn2+. Lysates were immunoblotted with antibodies against pAkt2S474. (F–H, bottom panels) Percentage of pAkt2S474 (% relative density units [RDU]) in neutrophils. UT cell band density was considered 0%; band density of fMLP-treated cells subtracted from UT band density was set at 100%. Data in panels D–I represent mean ± SEM of 4 or more experiments. *P < 0.0001, 1-way ANOVA.