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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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Rac1 modulates TLR-dependent leukocyte adhesion via ROS production. (A) ROS levels in THP-1 cells pretreated or not with a Rac1 inhibitor and subsequently stimulated with HKLM (TLR2 ligand) were assessed by flow cytometry using CellROX green reagent. Data are expressed as relative MFI compared with untreated THP-1 cells. Data are shown as mean ± SEM (n = 3 or 4 independent experiments). *p < 0.05. Student's t test was used for the statistical analysis. (B) Adhesion of THP-1 cells pretreated with dimethyl sulfoxide (Ctrl) or Rac1 inhibitor to immobilize ICAM-1 is shown in the absence or presence of indicated TLR ligands. Data expressed as relative adhesion. One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with control-treated unstimulated cells. Data are the mean ± SEM (n = 3 independent experiments).
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Figure 5: Rac1 modulates TLR-dependent leukocyte adhesion via ROS production. (A) ROS levels in THP-1 cells pretreated or not with a Rac1 inhibitor and subsequently stimulated with HKLM (TLR2 ligand) were assessed by flow cytometry using CellROX green reagent. Data are expressed as relative MFI compared with untreated THP-1 cells. Data are shown as mean ± SEM (n = 3 or 4 independent experiments). *p < 0.05. Student's t test was used for the statistical analysis. (B) Adhesion of THP-1 cells pretreated with dimethyl sulfoxide (Ctrl) or Rac1 inhibitor to immobilize ICAM-1 is shown in the absence or presence of indicated TLR ligands. Data expressed as relative adhesion. One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with control-treated unstimulated cells. Data are the mean ± SEM (n = 3 independent experiments).

Mentions: Rac1 is an intermediate in TLR-induced signaling (Shen et al., 2010) and ROS activation (Bäumer et al., 2008). We therefore studied the role of Rac1 in the TLR-dependent pathway of β2-integrin activation. We initially observed that treatment with a Rac1 inhibitor significantly down-regulated TLR2-induced ROS production (Figure 5A). Moreover, Rac1 inhibition abolished THP-1 myelomonocyte adhesion to ICAM-1 after TLR2 and TLR5 stimulation (Figure 5B). Taken together, TLR2- and TLR5-dependent activation of Rap1 GTPase and consequent β2-integrin affinity and β2-integrin–dependent leukocyte adhesion required the activation of Rac1 and Nox2 upon TLR2 and TLR5 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)

Rac1 modulates TLR-dependent leukocyte adhesion via ROS production. (A) ROS levels in THP-1 cells pretreated or not with a Rac1 inhibitor and subsequently stimulated with HKLM (TLR2 ligand) were assessed by flow cytometry using CellROX green reagent. Data are expressed as relative MFI compared with untreated THP-1 cells. Data are shown as mean ± SEM (n = 3 or 4 independent experiments). *p < 0.05. Student's t test was used for the statistical analysis. (B) Adhesion of THP-1 cells pretreated with dimethyl sulfoxide (Ctrl) or Rac1 inhibitor to immobilize ICAM-1 is shown in the absence or presence of indicated TLR ligands. Data expressed as relative adhesion. One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with control-treated unstimulated cells. Data are the mean ± SEM (n = 3 independent experiments).
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Related In: Results  -  Collection

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Figure 5: Rac1 modulates TLR-dependent leukocyte adhesion via ROS production. (A) ROS levels in THP-1 cells pretreated or not with a Rac1 inhibitor and subsequently stimulated with HKLM (TLR2 ligand) were assessed by flow cytometry using CellROX green reagent. Data are expressed as relative MFI compared with untreated THP-1 cells. Data are shown as mean ± SEM (n = 3 or 4 independent experiments). *p < 0.05. Student's t test was used for the statistical analysis. (B) Adhesion of THP-1 cells pretreated with dimethyl sulfoxide (Ctrl) or Rac1 inhibitor to immobilize ICAM-1 is shown in the absence or presence of indicated TLR ligands. Data expressed as relative adhesion. One-way ANOVA with Bonferroni posthoc analysis (asterisk and section sign [§] denote significance of the posthoc test; the latter symbol indicates comparison with control-treated unstimulated cells. Data are the mean ± SEM (n = 3 independent experiments).
Mentions: Rac1 is an intermediate in TLR-induced signaling (Shen et al., 2010) and ROS activation (Bäumer et al., 2008). We therefore studied the role of Rac1 in the TLR-dependent pathway of β2-integrin activation. We initially observed that treatment with a Rac1 inhibitor significantly down-regulated TLR2-induced ROS production (Figure 5A). Moreover, Rac1 inhibition abolished THP-1 myelomonocyte adhesion to ICAM-1 after TLR2 and TLR5 stimulation (Figure 5B). Taken together, TLR2- and TLR5-dependent activation of Rap1 GTPase and consequent β2-integrin affinity and β2-integrin–dependent leukocyte adhesion required the activation of Rac1 and Nox2 upon TLR2 and TLR5 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.

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Related in: MedlinePlus