Limits...
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.

Show MeSH

Related in: MedlinePlus

TLR2 and TLR5 stimulation activates β2-integrin affinity in a pathway that involves Rap1-GTPase. (A, B) To detect activation-dependent neoepitopes on β2-integrins, THP-1 myelomonocytes were stimulated without or with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand). The conformational status of β2-intergrin was studied by flow cytometry using Kim127 and mAb24 antibodies. The total surface expression of β2-intergrin was analyzed by an anti-CD18 antibody. (A) Representative histograms of fluorescence intensity and (B) relative mean fluorescence intensity (MFI) compared with untreated THP-1 cells (n = 3–5 per group). *p < 0.05. Student's t test was used for statistical analysis. (C) Rap1-GTP (activated Rap1) levels in THP-1 myelomonocytes treated or not with TLR2 ligand, TLR5 ligand, or TLR9 ligand for 10 min. Total Rap1 protein levels were used as loading control. Data derived from one representative experiment. (D) Densitometric analysis of immunoblots indicating activation of Rap1 by TLR2 and TLR5 ligation (n = 5–7 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (E) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Rap1a to immobilized ICAM-1 in the absence or presence of the indicated 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 unstimulated cells transfected with control siRNA; n = 3 independent experiments). (F) β2-Integrin conformational status in THP-1 cells transfected with siRNA targeting Rap1a in the absence or presence of HKLM (TLR2 ligand) or flagellin (TLR5 ligand) was assessed by flow cytometry using mAb24. Data expressed as relative MFI compared with unstimulated cells (n = 4 independent experiments). Data are shown as mean ± SEM. *p < 0.05. Student's t test was used for statistical analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230584&req=5

Figure 3: TLR2 and TLR5 stimulation activates β2-integrin affinity in a pathway that involves Rap1-GTPase. (A, B) To detect activation-dependent neoepitopes on β2-integrins, THP-1 myelomonocytes were stimulated without or with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand). The conformational status of β2-intergrin was studied by flow cytometry using Kim127 and mAb24 antibodies. The total surface expression of β2-intergrin was analyzed by an anti-CD18 antibody. (A) Representative histograms of fluorescence intensity and (B) relative mean fluorescence intensity (MFI) compared with untreated THP-1 cells (n = 3–5 per group). *p < 0.05. Student's t test was used for statistical analysis. (C) Rap1-GTP (activated Rap1) levels in THP-1 myelomonocytes treated or not with TLR2 ligand, TLR5 ligand, or TLR9 ligand for 10 min. Total Rap1 protein levels were used as loading control. Data derived from one representative experiment. (D) Densitometric analysis of immunoblots indicating activation of Rap1 by TLR2 and TLR5 ligation (n = 5–7 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (E) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Rap1a to immobilized ICAM-1 in the absence or presence of the indicated 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 unstimulated cells transfected with control siRNA; n = 3 independent experiments). (F) β2-Integrin conformational status in THP-1 cells transfected with siRNA targeting Rap1a in the absence or presence of HKLM (TLR2 ligand) or flagellin (TLR5 ligand) was assessed by flow cytometry using mAb24. Data expressed as relative MFI compared with unstimulated cells (n = 4 independent experiments). Data are shown as mean ± SEM. *p < 0.05. Student's t test was used for statistical analysis.

Mentions: The rapid activation of leukocyte adhesion in vitro by TLR2 and TLR5 and in vivo by TLR2 ligation implied alterations in β2-integrin affinity rather than changes in their expression upon TLR2 and TLR5 ligation. To this end, we stimulated THP-1 myelomonocytes acutely (20 min) with TLR2 (HKLM) or TLR5 (flagellin) ligands, used TLR9 (ODN2006) ligand as a negative control, and assessed β2-integrin conformational status by flow cytometry using KIM127 and mAb24 antibodies, which recognize the intermediate- and high-affinity conformations on β2-integrins (Stanley et al., 2008; Kuwano et al., 2010). Acute TLR2 and TLR5 activation increased the expression of the KIM127- and mAb24-recognized neoepitopes on β2-integrins (Figure 3, A and B) without affecting β2-integrin expression. In contrast, TLR9 stimulation had no effect on the conformational status of β2-integrins (Figure 3, A and B). Additional experiments demonstrated that the activation of the conformational status of β2-integrins by TLR2 stimulation can be detected as early as 1 min upon treatment with the ligand (Supplemental Figure S2A). Thus TLR2 and TLR5 ligation can rapidly induce the intermediate- and high-affinity conformation of β2-integrins.


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)

TLR2 and TLR5 stimulation activates β2-integrin affinity in a pathway that involves Rap1-GTPase. (A, B) To detect activation-dependent neoepitopes on β2-integrins, THP-1 myelomonocytes were stimulated without or with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand). The conformational status of β2-intergrin was studied by flow cytometry using Kim127 and mAb24 antibodies. The total surface expression of β2-intergrin was analyzed by an anti-CD18 antibody. (A) Representative histograms of fluorescence intensity and (B) relative mean fluorescence intensity (MFI) compared with untreated THP-1 cells (n = 3–5 per group). *p < 0.05. Student's t test was used for statistical analysis. (C) Rap1-GTP (activated Rap1) levels in THP-1 myelomonocytes treated or not with TLR2 ligand, TLR5 ligand, or TLR9 ligand for 10 min. Total Rap1 protein levels were used as loading control. Data derived from one representative experiment. (D) Densitometric analysis of immunoblots indicating activation of Rap1 by TLR2 and TLR5 ligation (n = 5–7 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (E) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Rap1a to immobilized ICAM-1 in the absence or presence of the indicated 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 unstimulated cells transfected with control siRNA; n = 3 independent experiments). (F) β2-Integrin conformational status in THP-1 cells transfected with siRNA targeting Rap1a in the absence or presence of HKLM (TLR2 ligand) or flagellin (TLR5 ligand) was assessed by flow cytometry using mAb24. Data expressed as relative MFI compared with unstimulated cells (n = 4 independent experiments). Data are shown as mean ± SEM. *p < 0.05. Student's t test was used for statistical analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230584&req=5

Figure 3: TLR2 and TLR5 stimulation activates β2-integrin affinity in a pathway that involves Rap1-GTPase. (A, B) To detect activation-dependent neoepitopes on β2-integrins, THP-1 myelomonocytes were stimulated without or with HKLM (TLR2 ligand), flagellin (TLR5 ligand), or ODN2006 (TLR9 ligand). The conformational status of β2-intergrin was studied by flow cytometry using Kim127 and mAb24 antibodies. The total surface expression of β2-intergrin was analyzed by an anti-CD18 antibody. (A) Representative histograms of fluorescence intensity and (B) relative mean fluorescence intensity (MFI) compared with untreated THP-1 cells (n = 3–5 per group). *p < 0.05. Student's t test was used for statistical analysis. (C) Rap1-GTP (activated Rap1) levels in THP-1 myelomonocytes treated or not with TLR2 ligand, TLR5 ligand, or TLR9 ligand for 10 min. Total Rap1 protein levels were used as loading control. Data derived from one representative experiment. (D) Densitometric analysis of immunoblots indicating activation of Rap1 by TLR2 and TLR5 ligation (n = 5–7 independent experiments). *p < 0.05. Student's t test was used for statistical analysis. (E) Adhesion of THP-1 cells transfected with control siRNA (Mock) or siRNA targeting Rap1a to immobilized ICAM-1 in the absence or presence of the indicated 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 unstimulated cells transfected with control siRNA; n = 3 independent experiments). (F) β2-Integrin conformational status in THP-1 cells transfected with siRNA targeting Rap1a in the absence or presence of HKLM (TLR2 ligand) or flagellin (TLR5 ligand) was assessed by flow cytometry using mAb24. Data expressed as relative MFI compared with unstimulated cells (n = 4 independent experiments). Data are shown as mean ± SEM. *p < 0.05. Student's t test was used for statistical analysis.
Mentions: The rapid activation of leukocyte adhesion in vitro by TLR2 and TLR5 and in vivo by TLR2 ligation implied alterations in β2-integrin affinity rather than changes in their expression upon TLR2 and TLR5 ligation. To this end, we stimulated THP-1 myelomonocytes acutely (20 min) with TLR2 (HKLM) or TLR5 (flagellin) ligands, used TLR9 (ODN2006) ligand as a negative control, and assessed β2-integrin conformational status by flow cytometry using KIM127 and mAb24 antibodies, which recognize the intermediate- and high-affinity conformations on β2-integrins (Stanley et al., 2008; Kuwano et al., 2010). Acute TLR2 and TLR5 activation increased the expression of the KIM127- and mAb24-recognized neoepitopes on β2-integrins (Figure 3, A and B) without affecting β2-integrin expression. In contrast, TLR9 stimulation had no effect on the conformational status of β2-integrins (Figure 3, A and B). Additional experiments demonstrated that the activation of the conformational status of β2-integrins by TLR2 stimulation can be detected as early as 1 min upon treatment with the ligand (Supplemental Figure S2A). Thus TLR2 and TLR5 ligation can rapidly induce the intermediate- and high-affinity conformation of β2-integrins.

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