Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating beta2 integrins - PubMed (original) (raw)

Nonmuscle myosin light-chain kinase mediates neutrophil transmigration in sepsis-induced lung inflammation by activating beta2 integrins

Jingsong Xu et al. Nat Immunol. 2008 Aug.

Abstract

Nonmuscle myosin light-chain kinase (MYLK) mediates increased lung vascular endothelial permeability in lipopolysaccharide-induced lung inflammatory injury, the chief cause of the acute respiratory distress syndrome. In a lung injury model, we demonstrate here that MYLK was also essential for neutrophil transmigration, but that this function was mostly independent of myosin II regulatory light chain, the only known substrate of MYLK. Instead, MYLK in neutrophils was required for the recruitment and activation of the tyrosine kinase Pyk2, which mediated full activation of beta(2) integrins. Our results demonstrate that MYLK-mediated activation of beta(2) integrins through Pyk2 links beta(2) integrin signaling to the actin motile machinery of neutrophils.

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Figures

Figure 1

Figure 1. Ex vivo LPS-induced lung injury and edema formation

(a) Change in microvessel permeability in wild-type lungs after perfusion of wild-type (WT) or _Mylk_−/− neutrophils (PMN), followed by challenge for 16 h with LPS (10 mg/kg given intraperitoneally), assessed as the pulmonary microvessel filtration coefficient (_K_fc) and presented as per gram of dry lung weight (dry g). (b) Time-dependent change in pulmonary edema formation, as assessed by the increase in wet weight of lungs treated as described in a. (c) Sequestration of 111In-oxine–labeled PMNs in lungs after 111In-labeled PMNs were stimulated with LPS or fMLP and then added to perfusates of lung preparations. (d) Adhesion of wild-type and _Mylk_−/− PMNs to cultured mouse lung vascular endothelial cells. (e) Transmigration of LPS-primed wild-type and _Mylk_−/− PMNs across endothelial cells toward fMLP in the lower chamber of a Transwell. (f) Migration of PMNs into the airspace of wild-type and _Mylk_−/− mice challenged with LPS. *, P < 0.05, compared with basal; **, P < 0.05, compared with wild type after LPS stimulation. Data are the mean of five (a,b), four (c–e) or six (f) independent experiments (error bars, s.e.m.).

Figure 2

Figure 2. Loss of MYLK function fails to prevent myosin II activation

(a,b) MLC phosphorylation in wild-type and _Mylk_−/− lungs (a) or PMNs (b). Bottom blots, confirmation of equal protein loading by analysis with anti-MLC. Above, densitometry analysis. *, P < 0.05, compared with basal (no LPS stimulation). Data are from one experiment representative of three (error bars, s.e.m.). (c) Subcellular distribution of activated myosin II (top; anti-p19-MLC) and MYLK (bottom; anti-MYLK) in polarized PMNs. Arrows indicate leading edges. Scale bars, 10 µm. Results are representative of three experiments. (d) Adhesion of wild-type PMNs preincubated with LPS (1 µg/ml), then cultured for 30 min at 37 °C together with mouse lung vascular endothelial cells with or without inhibitors (horizontal axis). Blebbi, blebbistatin. *, P < 0.05, compared with basal; **, P < 0.05, treatment with inhibitor plus LPS compared with LPS alone. Data are the mean of four independent experiments (error bars, s.e.m.).

Figure 3

Figure 3. Activation of β2 integrin in neutrophils

(a) Microscopy of wild-type and _Mylk_−/− PMNs spread on ICAM-1–coated surfaces, then fixed with methanol and stained with anti-aM. Original magnification, ×60. (b) Adhesion to ICAM-1–coated surfaces of wild-type and _Mylk_−/− PMNs challenged with LPS or not. *, P < 0.05, compared with no LPS; **, P < 0.05, compared with wild type after LPS stimulation. (c) Analysis of β2 integrin activation by soluble ICAM-1 binding assay with (+) or without (−) 10 mM Mn2+. ICAM-1–Fc, Fc-tagged ICAM-1. *, P < 0.05, compared with no stimulation; **, P < 0.05, compared with wild type after Mn2+ stimulation. (d) Flow cytometry of the surface expression of β2 integrins on circulating wild-type and _Mylk_−/− PMNs. *, P < 0.05, compared with no LPS. Data are from one experiment representative of three (a) or are the mean of four independent experiments (b–d; error bars, s.e.m.).

Figure 4

Figure 4. Tyrosine kinase activation and interaction with β2 integrin

(a) Phosphorylation (p-) of c-Src, Syk and Pyk2 in wild-type and _Mylk_−/− PMNs. Bottom blots, confirmation of equal protein loading by analysis with anti-c-Src, anti-Pyk2 and anti-Syk. Above, densitometry analysis. *, P < 0.05, compared with no LPS; **, P < 0.05, compared with other groups after LPS stimulation. Data are the mean of four independent experiments (error bars, s.e.m.). (b) Colocalization of MYLK and Pyk2 in wild-type PMNs stimulated with fMLP and stained with anti-MYLK and anti-Pyk2 (above). Arrows indicate leading edges. Scale bar, 10 µm. Below, coimmunoprecipitation of MYLK and Pyk2 in wild-type PMNs. IP, immunoprecipitation; IB, immunoblot. Data are from one experiment representative of three. (c) Association of β2 integrin with c-Src and Pyk2 in wild-type and _Mylk_−/− PMNs stimulated for 0, 30 or 60 min with LPS (1 µg/ml); equal amounts of protein immunoprecipitated from lysates with anti–β2 integrin are analyzed by immunoblot with anti-Pyk2, anti-c-Src or anti–β2 integrin. IgG, immunoglobulin G (control antibody). Data are representative of three independent experiments.

Figure 5

Figure 5. The interaction of β2 integrin and the cytoskeleton in neutrophils

Association of β2 integrin with the actin-associated proteins talin (a) and α-actinin (b) in wild-type and _Mylk_−/− PMNs stimulated for 0, 30 or 60 min with LPS (1 µg/ml); equal amounts of protein immunoprecipitated from lysates with anti–β2 integrin are analyzed by immunoblot with anti-talin or anti-α-actinin. IgG, control antibody. Data are representative of three independent experiments.

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References

    1. Cohen MS. Molecular events in the activation of human neutrophils for microbial killing. Clin. Infect. Dis. 1994;18 Suppl 2:S170–S179. - PubMed
    1. Stevens T, Garcia JG, Shasby DM, Bhattacharya J, Malik AB. Mechanisms regulating endothelial cell barrier function. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000;279:L419–L422. - PubMed
    1. Liu Y, et al. Regulation of leukocyte transmigration: cell surface interactions and signaling events. J. Immunol. 2004;172:7–13. - PubMed
    1. Simpson SQ, Casey LC. Role of tumor necrosis factor in sepsis and acute lung injury. Crit. Care Clin. 1989;5:27–47. - PubMed
    1. Crockett-Torabi E, Ward PA. The role of leukocytes in tissue injury. Eur. J. Anaesthesiol. 1996;13:235–246. - PubMed

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