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Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE.

Moreira-Tabaka H, Peluso J, Vonesch JL, Hentsch D, Kessler P, Reimund JM, Dumont S, Muller CD - PLoS ONE (2012)

Bottom Line: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α.Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE.This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France. heltab@basmed.am.wroc.pl

ABSTRACT

Background: Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.

Methodology/principal findings: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.

Conclusions/significance: On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.

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THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment.Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p<0.05).
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pone-0034184-g002: THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment.Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p<0.05).

Mentions: TNF-α is initially synthesized as a transmembrane precursor (m-TNF-α), then processed by TACE and/or other sheddase to become a secreted soluble form. Blocking of this process e.g. by TACE inhibitors should thus generate TNF-α retention and accumulation on the cellular membrane. To verify the cogency of our hypothesis, we evaluated m-TNF-α expression in cells pretreated by Ro 32-7315, a specific TACE inhibitor. As demonstrated by flow cytometry (Figure 2 panel A and Figure 3), Ro 32-7315 treatment yielded in an important accumulation of m-TNF-α. The obtained MFI (AU) values for THP-1 cells and PBMC were 260 vs. 64 (p = 0.049) and 90 vs. 30 (p = 0.046) (treated vs. control cells), respectively. We further performed confocal microscopy in order to validate the membrane localization of retained TNF-α after TACE inhibitor treatment. As shown in Figure 4, a strong cell surface staining was visible in the presence of Ro 32-7315 in both THP-1 and monocytes. Visualization of membrane anchored TNF-α revealed a punctuated distribution at the cell surface, suggesting a lipid raft localization of TNF-α. Tellier et al. have shown that the mature form of TNF-α is mainly present in the cholesterol-rich membrane microdomains (lipid rafts) and that an inhibition of metalloproteases increases the proportion of TNF-α in those parts [31]. Thus our results confirm that inhibiting TACE activity leads to an accumulation of unprocessed TNF-α on the cell surface.


Unlike for human monocytes after LPS activation, release of TNF-α by THP-1 cells is produced by a TACE catalytically different from constitutive TACE.

Moreira-Tabaka H, Peluso J, Vonesch JL, Hentsch D, Kessler P, Reimund JM, Dumont S, Muller CD - PLoS ONE (2012)

THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment.Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316627&req=5

pone-0034184-g002: THP-1 cell surface accumulation of membrane-bound TNF-α after Ro 32-7315 and TQ (Panel A) or Celastrol (Panel B) treatment.Cells were stimulated for 4 hours (1 µg/ml of LPS) in the presence of Ro 32-7315 (3.5 µM), TQ (10 µM) and Celastrol (10 µM) or 0.1% (v/v) DMSO as a control. The intact cells were then stained by FITC-anti-m-TNF-α MAb. Membrane-bound TNF-α expression was evaluated in flow cytometry. Representative histograms are shown. The fluorescence intensity (AU) is plotted versus the number of cells. For all 3 compounds, membrane –bound TNF-α expression increased after THP-1 cells were treated (p<0.05).
Mentions: TNF-α is initially synthesized as a transmembrane precursor (m-TNF-α), then processed by TACE and/or other sheddase to become a secreted soluble form. Blocking of this process e.g. by TACE inhibitors should thus generate TNF-α retention and accumulation on the cellular membrane. To verify the cogency of our hypothesis, we evaluated m-TNF-α expression in cells pretreated by Ro 32-7315, a specific TACE inhibitor. As demonstrated by flow cytometry (Figure 2 panel A and Figure 3), Ro 32-7315 treatment yielded in an important accumulation of m-TNF-α. The obtained MFI (AU) values for THP-1 cells and PBMC were 260 vs. 64 (p = 0.049) and 90 vs. 30 (p = 0.046) (treated vs. control cells), respectively. We further performed confocal microscopy in order to validate the membrane localization of retained TNF-α after TACE inhibitor treatment. As shown in Figure 4, a strong cell surface staining was visible in the presence of Ro 32-7315 in both THP-1 and monocytes. Visualization of membrane anchored TNF-α revealed a punctuated distribution at the cell surface, suggesting a lipid raft localization of TNF-α. Tellier et al. have shown that the mature form of TNF-α is mainly present in the cholesterol-rich membrane microdomains (lipid rafts) and that an inhibition of metalloproteases increases the proportion of TNF-α in those parts [31]. Thus our results confirm that inhibiting TACE activity leads to an accumulation of unprocessed TNF-α on the cell surface.

Bottom Line: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α.Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE.This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Innovation Thérapeutique, UMR 7200, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France. heltab@basmed.am.wroc.pl

ABSTRACT

Background: Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine today identified as a key mediator of several chronic inflammatory diseases. TNF-α, initially synthesized as a membrane-anchored precursor (pro-TNF-α), is processed by proteolytic cleavage to generate the secreted mature form. TNF-α converting enzyme (TACE) is currently the first and single protease described as responsible for the inducible release of soluble TNF-α.

Methodology/principal findings: Here, we demonstrated the presence on THP-1 cells as on human monocytes of a constitutive proteolytical activity able to cleave pro-TNF-α. Revelation of the cell surface TACE protein expression confirmed that the observed catalytic activity is due to TACE. However, further studies using effective and innovative TNF-α inhibitors, as well as a highly selective TACE inhibitor, support the presence of a catalytically different sheddase activity on LPS activated THP-1 cells. It appears that this catalytically different TACE protease activity might have a significant contribution to TNF-α release in LPS activated THP-1 cells, by contrast to human monocytes where the TACE activity remains catalytically unchanged even after LPS activation.

Conclusions/significance: On the surface of LPS activated THP-1 cells we identified a releasing TNF-α activity, catalytically different from the sheddase activity observed on human monocytes from healthy donors. This catalytically-modified TACE activity is different from the constitutive shedding activity and appears only upon stimulation by LPS.

Show MeSH
Related in: MedlinePlus