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A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.

Chung KJ, Mitroulis I, Wiessner JR, Zheng YY, Siegert G, Sperandio M, Chavakis T - Mol. Biol. Cell (2014)

Bottom Line: Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model.TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes.This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany kyoung-jin.chung@uniklinikum-dresden.de.

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Adhesion of inflammatory cells in vivo in the cremaster muscle model is enhanced by Pam3CSK4 administration. (A) Leukocyte adhesion efficiency (percentage of adherent leukocytes per square millimeter relative to the white blood cell count per microliter), (B) number of adherent leukocytes per vessel surface (in square millimeters), (C) leukocyte rolling flux fraction (percentage of rolling leukocytes relative to the number of leukocytes entering the vessel), and (D) leukocyte rolling velocities investigated in exteriorized cremaster muscle venules at baseline conditions (before PAM), as well as 1–2 min after Pam3CSK4 injection (after Pam). Results are shown as mean ± SEM in A–C (*p < 0.05, n = 9 mice). Leukocyte rolling velocities in D are displayed as cumulative histogram of 82 analyzed leukocyte rolling velocities before and 83 measured rolling velocities 1–2 min after Pam3CSK4 injection. Student's t test was used for statistical analysis.
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Figure 2: Adhesion of inflammatory cells in vivo in the cremaster muscle model is enhanced by Pam3CSK4 administration. (A) Leukocyte adhesion efficiency (percentage of adherent leukocytes per square millimeter relative to the white blood cell count per microliter), (B) number of adherent leukocytes per vessel surface (in square millimeters), (C) leukocyte rolling flux fraction (percentage of rolling leukocytes relative to the number of leukocytes entering the vessel), and (D) leukocyte rolling velocities investigated in exteriorized cremaster muscle venules at baseline conditions (before PAM), as well as 1–2 min after Pam3CSK4 injection (after Pam). Results are shown as mean ± SEM in A–C (*p < 0.05, n = 9 mice). Leukocyte rolling velocities in D are displayed as cumulative histogram of 82 analyzed leukocyte rolling velocities before and 83 measured rolling velocities 1–2 min after Pam3CSK4 injection. Student's t test was used for statistical analysis.

Mentions: We next tested whether the observed stimulation of leukocyte adhesion by TLR ligation in vitro could be relevant in vivo as well. To address acute leukocyte adhesion in vivo, we used the cremaster model of acute inflammation associated with intravital microscopy analysis. We tested whether systemic injection of the TLR2-agonist Pam3CSK4 into C57BL/6 mice via a carotid artery catheter led to induction of firm leukocyte arrest in exteriorized cremaster muscle venules in vivo. We found that after the first minute postinjection of the TLR2 ligand, leukocyte adhesion efficiency (number of adherent leukocytes/systemic leukocyte count) was significantly elevated compared with leukocyte adhesion efficiency before injection of Pam3CSK4 (Figure 2A). Similarly, the absolute number of adherent leukocytes 1–2 min after Pam3CSK4 injection was significantly higher than the number before Pam3CSK4 injection (Figure 2B and Supplemental Videos S1 and S2), suggesting that signaling via TLR2 can induce rapid firm leukocyte arrest in vivo. We found no significant changes in the leukocyte rolling flux fraction before and 1–2 min after injection of the TLR2 agonist Pam3CSK4 (Figure 2C). In contrast, leukocyte rolling velocities decreased significantly upon stimulation with Pam3CSK4 (Figure 2D), implying that TLR2 ligation induced a transition from rolling to firm leukocyte adhesion. In contrast, systemic injection of the TLR9 agonist ODN1668 had no effect on leukocyte adhesion to endothelial cells (Supplemental Figure S1). Besides the well-established dependence of firm leukocyte arrest on leukocyte β2-integrins (Henderson et al., 2001), previous work also suggested that the transition from rolling to firm leukocyte adhesion is accompanied by intermediate activation of the β2-integrin LFA-1, which has been linked to a reduction in leukocyte rolling velocities in vivo (Zarbock et al., 2008). Taken together, our in vivo findings, supported by our in vitro data, point to a previously unknown rapid activation of β2-integrin–dependent leukocyte adhesion upon TLR2 ligation.


A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase.

Chung KJ, Mitroulis I, Wiessner JR, Zheng YY, Siegert G, Sperandio M, Chavakis T - Mol. Biol. Cell (2014)

Adhesion of inflammatory cells in vivo in the cremaster muscle model is enhanced by Pam3CSK4 administration. (A) Leukocyte adhesion efficiency (percentage of adherent leukocytes per square millimeter relative to the white blood cell count per microliter), (B) number of adherent leukocytes per vessel surface (in square millimeters), (C) leukocyte rolling flux fraction (percentage of rolling leukocytes relative to the number of leukocytes entering the vessel), and (D) leukocyte rolling velocities investigated in exteriorized cremaster muscle venules at baseline conditions (before PAM), as well as 1–2 min after Pam3CSK4 injection (after Pam). Results are shown as mean ± SEM in A–C (*p < 0.05, n = 9 mice). Leukocyte rolling velocities in D are displayed as cumulative histogram of 82 analyzed leukocyte rolling velocities before and 83 measured rolling velocities 1–2 min after Pam3CSK4 injection. Student's t test was used for statistical analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Adhesion of inflammatory cells in vivo in the cremaster muscle model is enhanced by Pam3CSK4 administration. (A) Leukocyte adhesion efficiency (percentage of adherent leukocytes per square millimeter relative to the white blood cell count per microliter), (B) number of adherent leukocytes per vessel surface (in square millimeters), (C) leukocyte rolling flux fraction (percentage of rolling leukocytes relative to the number of leukocytes entering the vessel), and (D) leukocyte rolling velocities investigated in exteriorized cremaster muscle venules at baseline conditions (before PAM), as well as 1–2 min after Pam3CSK4 injection (after Pam). Results are shown as mean ± SEM in A–C (*p < 0.05, n = 9 mice). Leukocyte rolling velocities in D are displayed as cumulative histogram of 82 analyzed leukocyte rolling velocities before and 83 measured rolling velocities 1–2 min after Pam3CSK4 injection. Student's t test was used for statistical analysis.
Mentions: We next tested whether the observed stimulation of leukocyte adhesion by TLR ligation in vitro could be relevant in vivo as well. To address acute leukocyte adhesion in vivo, we used the cremaster model of acute inflammation associated with intravital microscopy analysis. We tested whether systemic injection of the TLR2-agonist Pam3CSK4 into C57BL/6 mice via a carotid artery catheter led to induction of firm leukocyte arrest in exteriorized cremaster muscle venules in vivo. We found that after the first minute postinjection of the TLR2 ligand, leukocyte adhesion efficiency (number of adherent leukocytes/systemic leukocyte count) was significantly elevated compared with leukocyte adhesion efficiency before injection of Pam3CSK4 (Figure 2A). Similarly, the absolute number of adherent leukocytes 1–2 min after Pam3CSK4 injection was significantly higher than the number before Pam3CSK4 injection (Figure 2B and Supplemental Videos S1 and S2), suggesting that signaling via TLR2 can induce rapid firm leukocyte arrest in vivo. We found no significant changes in the leukocyte rolling flux fraction before and 1–2 min after injection of the TLR2 agonist Pam3CSK4 (Figure 2C). In contrast, leukocyte rolling velocities decreased significantly upon stimulation with Pam3CSK4 (Figure 2D), implying that TLR2 ligation induced a transition from rolling to firm leukocyte adhesion. In contrast, systemic injection of the TLR9 agonist ODN1668 had no effect on leukocyte adhesion to endothelial cells (Supplemental Figure S1). Besides the well-established dependence of firm leukocyte arrest on leukocyte β2-integrins (Henderson et al., 2001), previous work also suggested that the transition from rolling to firm leukocyte adhesion is accompanied by intermediate activation of the β2-integrin LFA-1, which has been linked to a reduction in leukocyte rolling velocities in vivo (Zarbock et al., 2008). Taken together, our in vivo findings, supported by our in vitro data, point to a previously unknown rapid activation of β2-integrin–dependent leukocyte adhesion upon TLR2 ligation.

Bottom Line: Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model.TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes.This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pathobiochemistry, Technische Universität Dresden, 01309 Dresden, Germany Institute of Physiology, Technische Universität Dresden, 01309 Dresden, Germany kyoung-jin.chung@uniklinikum-dresden.de.

Show MeSH
Related in: MedlinePlus