Figure 6.
Local injection of TNF-α or inactivated thrombin induces NET-dependent intralymphatic fibrin coagulation and blocks lymphatic drainage in mouse skin. (A) Representative immunofluorescence images of thrombomodulin (thrmod, red), CD31 (green), tissue factor (TF, green), and von Willebrand factor (von Willnbrd, red) staining in mouse ears. Arrows point to collecting lymphatics that contain unique morphological features (ie, valves with uneven vessel diameters). Thrombomodulin expression overlaps with CD31 and is also present in some interstitial and perivascular cells and nerve fibers (left) (n). Both blood and lymphatic vessels express thrombomodulin, but only blood vessels express TF (middle). TF expression in lymphatics is limited to perivascular cells and adipocytes (A). In contrast to blood vessels, lymphatic vessels did not express von Willebrand factor (right). Scale bars, 50 μm. (B) Schematic of experimental design: intradermal injection of clot initiators (CIs) TNF-α, TNF-α and IL-1β, or inactivated thrombin, on day 0 was followed by IV injection of FITC-labeled fibrinogen 30 minutes later. Six hours after the first injection, the procedure was repeated. The mice were perfused and fixed after 20 hours. (C) Representative confocal whole-mount images of lymphatic collectors occluded by fibrin (green) clots after treatment with TNF-α (left), TNF-α and IL-1β (middle), or inactivated thrombin (right). Basement membrane (BM, gray) is represented by collagen-IV staining. Scale bars, 50 μm. (D) Schematic of experimental design: intradermal injection of clot initiator (TNF-α) and intraperitoneal injection of DNase I on day 0 was followed by IV injection of 647-labeled fibrinogen 30 minutes later. Six hours after the first injection, the procedure was repeated, and the mice were perfused and fixed after 20 hours. Control mice received TNF-α but not DNase I injections. (E) Representative whole-mount immunofluorescence staining images of lymphatic vessels and clots in the mouse ear in control mice (left) and mice that received DNase I injections (right) (LYVE-1, white; H3cit, red; fibrinogen, green). White arrows indicate lymphatic clots (left inset: LYVE-1 and H3cit channels of clot containing NETs; right inset: LYVE-1 and H3cit channels of clot outside of a lymphatic with no NETs), and yellow arrows indicate clots outside of lymphatics, likely in a blood vessel. Scale bar, 100 μm. (F) Averaged number of lymphatic clots in the left and right ear dorsal dermis for each mouse injected intradermally with TNF-α (n = 5) and each mouse injected with both TNF-α and DNase I (n = 5). Bars and error bars represent median and 95% confidence interval, respectively. ∗∗∗P < .001 by unpaired two-tailed t test.

Local injection of TNF-α or inactivated thrombin induces NET-dependent intralymphatic fibrin coagulation and blocks lymphatic drainage in mouse skin. (A) Representative immunofluorescence images of thrombomodulin (thrmod, red), CD31 (green), tissue factor (TF, green), and von Willebrand factor (von Willnbrd, red) staining in mouse ears. Arrows point to collecting lymphatics that contain unique morphological features (ie, valves with uneven vessel diameters). Thrombomodulin expression overlaps with CD31 and is also present in some interstitial and perivascular cells and nerve fibers (left) (n). Both blood and lymphatic vessels express thrombomodulin, but only blood vessels express TF (middle). TF expression in lymphatics is limited to perivascular cells and adipocytes (A). In contrast to blood vessels, lymphatic vessels did not express von Willebrand factor (right). Scale bars, 50 μm. (B) Schematic of experimental design: intradermal injection of clot initiators (CIs) TNF-α, TNF-α and IL-1β, or inactivated thrombin, on day 0 was followed by IV injection of FITC-labeled fibrinogen 30 minutes later. Six hours after the first injection, the procedure was repeated. The mice were perfused and fixed after 20 hours. (C) Representative confocal whole-mount images of lymphatic collectors occluded by fibrin (green) clots after treatment with TNF-α (left), TNF-α and IL-1β (middle), or inactivated thrombin (right). Basement membrane (BM, gray) is represented by collagen-IV staining. Scale bars, 50 μm. (D) Schematic of experimental design: intradermal injection of clot initiator (TNF-α) and intraperitoneal injection of DNase I on day 0 was followed by IV injection of 647-labeled fibrinogen 30 minutes later. Six hours after the first injection, the procedure was repeated, and the mice were perfused and fixed after 20 hours. Control mice received TNF-α but not DNase I injections. (E) Representative whole-mount immunofluorescence staining images of lymphatic vessels and clots in the mouse ear in control mice (left) and mice that received DNase I injections (right) (LYVE-1, white; H3cit, red; fibrinogen, green). White arrows indicate lymphatic clots (left inset: LYVE-1 and H3cit channels of clot containing NETs; right inset: LYVE-1 and H3cit channels of clot outside of a lymphatic with no NETs), and yellow arrows indicate clots outside of lymphatics, likely in a blood vessel. Scale bar, 100 μm. (F) Averaged number of lymphatic clots in the left and right ear dorsal dermis for each mouse injected intradermally with TNF-α (n = 5) and each mouse injected with both TNF-α and DNase I (n = 5). Bars and error bars represent median and 95% confidence interval, respectively. ∗∗∗P < .001 by unpaired two-tailed t test.

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