MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane
Angela R.M. Kurz, Monika Pruenster, Ina Rohwedder, Mahalakshmi Ramadass, Kerstin Schäfer, Ute Harrison, Gabriel Gouveia, Claudia Nussbaum, Roland Immler, Johannes R. Wiessner, Andreas Margraf, Dae-Sik Lim, Barbara Walzog, Steffen Dietzel, Markus Moser, Christoph Klein, Dietmar Vestweber, Rainer Haas, Sergio D. Catz, Markus Sperandio
Angela R.M. Kurz, Monika Pruenster, Ina Rohwedder, Mahalakshmi Ramadass, Kerstin Schäfer, Ute Harrison, Gabriel Gouveia, Claudia Nussbaum, Roland Immler, Johannes R. Wiessner, Andreas Margraf, Dae-Sik Lim, Barbara Walzog, Steffen Dietzel, Markus Moser, Christoph Klein, Dietmar Vestweber, Rainer Haas, Sergio D. Catz, Markus Sperandio
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Research Article Inflammation

MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane

Abstract

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20–like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1–/–) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1–/– neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1–/– neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

Authors

Angela R.M. Kurz, Monika Pruenster, Ina Rohwedder, Mahalakshmi Ramadass, Kerstin Schäfer, Ute Harrison, Gabriel Gouveia, Claudia Nussbaum, Roland Immler, Johannes R. Wiessner, Andreas Margraf, Dae-Sik Lim, Barbara Walzog, Steffen Dietzel, Markus Moser, Christoph Klein, Dietmar Vestweber, Rainer Haas, Sergio D. Catz, Markus Sperandio

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Figure 5

MST1 is required for the mobilization of VLA-3, VLA-6, and NE to the plasma membrane.

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MST1 is required for the mobilization of VLA-3, VLA-6, and NE to the pla...
(A, D, and G) Immunostaining of representative WT and Mst1–/– neutrophil on BSA- or PECAM-1/ICAM-1/CXCL1–coated wells for VLA-3 (A), VLA-6 (D), and NE (G). Scale bar: 5 μm. (B, E, and H) Fluorescence intensity profiles along a line through the cell for VLA-3 (B), VLA-6 (E), and NE (H) of a representative WT (black line) and Mst1–/– neutrophil (gray line) seeded on PECAM-1/ICAM-1/CXCL1–coated wells. (C, F, and I) Quantification of ring formation of VLA-3 (C), VLA-6 (F), and NE (I) (n > 50 analyzed neutrophils from 3 mice per group, mean ± SEM, **P < 0.01, ***P < 0.001, 2-way ANOVA, Sidak’s multiple comparisons test or Tukey’s multiple comparisons test). (J) NE activity (white) within venules in unstimulated and TNF-α–stimulated cremaster muscle whole mounts from WT and Mst1–/– mice imaged by confocal microscopy. Venules were visualized using a rat anti–mouse PECAM-1 antibody (red) (n = 3 mice per group). Scale bar: 50 μm. (K) Total number of extravasated neutrophils in the peritoneal lavage 2 hours after i.p. injection of TNF-α in WT mice after treatment with either the VLA-3–blocking peptide LXY2, the α6-blocking mAb GoH3, and the NE inhibitor GW311616A; or solvent alone, control peptide, and isotype-matched control mAb (n = 6, mean ± SEM, **P < 0.01, unpaired t test).