Different signaling pathways stimulate a disintegrin and metalloprotease-17 (ADAM17) in neutrophils during apoptosis and activation

Abstract

ADAM17 is a membrane-associated metalloprotease that cleaves proteins from the surface of neutrophils and modulates the density of various receptors and adhesion molecules. The protease activity of ADAM17 is highly inducible and occurs upon neutrophil activation as well as apoptosis. At this time, little is known about the signal transduction pathway that promotes ADAM17 activity in neutrophils upon the induction of apoptosis. We show that caspase-8 activation, Bid cleavage, and the release of mitochondrial reactive oxygen species are sequential transduction components of the Fas signaling cascade that induces ADAM17. This is different from ADAM17 stimulation upon overt neutrophil activation, which requires MAPK p38 or ERK, but not caspases and reactive oxygen species. ADAM17 activity in apoptotic neutrophils may serve to inactivate select effector molecules that promote the pro-inflammatory activity of recruited neutrophils. For instance, TNFα receptors TNF-RI and TNF-RII are substrates of ADAM17, and we show that they are shed during apoptosis, decreasing neutrophil sensitivity to TNFα. Altogether, our findings provide significant new insights into the signal transduction pathway that stimulates ADAM17 during induced neutrophil apoptosis. ADAM17 induction during apoptosis may rapidly diminish neutrophil sensitivity to the inflammatory environment, complementing other anti-inflammatory activities by these cells during inflammation resolution.

FIGURE 1.

Fas-induced ADAM17 stimulation requires caspase-8, but not caspase-3. A, human peripheral blood neutrophils were either untreated or treated with the anti-Fas antibody CH-11 or fMLP for 6 h or 30 min, respectively, at 37 °C in the presence or absence of TAPI. Some neutrophils were initially incubated with a broad spectrum caspase inhibitor (z-VAD-fmk), a caspase-8 inhibitor (z-IETD-fmk), or a caspase-3 inhibitor (Ac-DMQD-cho) for 30 min at 25 °C, as indicated. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Shown is the mean (± S.D.) of at least three independent experiments. *, p < 0.05 versus CH-11 treatment alone. B, neutrophils were treated as described above, and detergent lysates from equivalent cell numbers were subjected to reducing SDS-PAGE and immunoblotting with an anti-caspase-3 mAb. The antibody detects full-length caspase-3 (Pro-caspase 3) and the large fragment of activated caspase-3 resulting from cleavage (17/19 kDa). Data are representative of at least three independent experiments using neutrophils isolated from separate donors.

FIGURE 2.

Jurkat cells model neutrophils for ADAM17 stimulation upon Fas signaling. A, Jurkat cells were either untreated or treated with the anti-Fas antibody CH-11 or rhFasL, as indicated, in the presence or absence of TAPI for 1.5 h at 37 °C. Some cells were initially incubated with the caspase inhibitors described in Fig. 1. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Results are expressed as mean (± S.D.) of at least three independent experiments. *, p < 0.05 versus CH-11 or FasL treatment alone. B, in addition, the cell pellets were stained for Annexin V-FITC to detect cell surface phosphatidylserine exposure. The x axis = log 10 fluorescence. Results are representative of three independent experiments.

FIGURE 3.

Bid knock-down abrogates ADAM17 stimulation upon Fas engagement. A, wild-type (WT), vector control, Bid-deficient (Bid shRNA), or Apaf-1-deficient (Apaf-1 shRNA) Jurkat cells were either untreated or treated with the anti-Fas antibody CH-11 for 1.5 h at 37 °C. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Results are expressed as mean (± S.D.) of at least three independent experiments. *, p < 0.001 versus wild-type Jurkat cells treated with CH-11. B, Jurkat cells were either untreated or treated with the anti-Fas antibody CH-11 for 1.5 h at 37 °C in the presence or absence of TAPI. Some cells were initially incubated with a caspase-9 inhibitor (Ac-LEHD-cmk) for 30 min at 25 °C, as indicated. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Shown is the mean (± S.D.) of at least three independent experiments. *, p < 0.05 versus CH-11 treatment alone. The cells were labeled with Annexin V-FITC and examined by flow cytometry. The x axis = log 10 fluorescence. Data are representative of at least three independent experiments.

FIGURE 4.

Mitochondrial ROS in neutrophils is important for Fas-induced ADAM17 stimulation. A, human peripheral blood neutrophils were either untreated or treated with rotenone for 1 h at 37 °C in the presence or absence of TAPI. Relative L-selectin or LFA-1 surface expression levels, as indicated, were determined by flow cytometry. Negative control Ab staining of untreated cells is indicated (dashed line). The x axis = log 10 fluorescence. Data are representative of at least three independent experiments using neutrophils isolated from separate donors. B, neutrophils were either untreated or treated with the anti-Fas antibody CH-11 for 6 h at 37 °C in the presence or absence of TAPI. Some neutrophils were initially incubated with the intracellular ROS scavenger NAC or the NADPH oxidase inhibitor DPI for 30 min at 25 °C, as indicated. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Results are expressed as mean (± S.D.) of at least three independent experiments. *, p < 0.01 versus CH-11 treatment alone.

FIGURE 5.

Fas-induced l-selectin shedding occurs independent of the p38 MAPK and ERK. Human peripheral blood neutrophils were either untreated or treated with the anti-Fas antibody CH-11 for 6 h at 37 °C in the presence or absence of TAPI. Some cells were initially incubated with a p38 inhibitor (SB203580) or an ERK pathway inhibitor (U0126) for 30 min at 25 °C, as indicated. Cell supernatant levels of soluble L-selectin were quantified by ELISA. Results are expressed as mean (± S.D.) of at least three independent experiments. *, p < 0.05 versus CH-11 treatment alone.

FIGURE 6.

Pro-protein to mature ADAM17 conversion is not increased during Fas-mediated apoptosis. Human peripheral blood neutrophils (A) or Jurkat cells (B) were either untreated or treated with the anti-Fas antibody CH-11 for 6 or 1.5 h, respectively, at 37 °C, as indicated. Detergent lysates from equivalent numbers of untreated and treated cells were then subjected to reducing SDS-PAGE and immunoblotting with antibodies to mature ADAM17, the pro-domain of ADAM17, or GAPDH (loading control), as shown in lanes 1 and 2. Detergent lysates of untreated cells were also immunoblotted with appropriate negative control antibodies (lane 3). Immunoblots are representative of three independent experiments. Densitometric data are from three separate experiments (expressed as mean ± S.D.) and shown as percentage of loading control.

FIGURE 7.

ADAM17 activity decreases the responsiveness of apoptotic neutrophils to TNFα. A, human peripheral blood neutrophils were either untreated or treated with the anti-Fas antibody CH-11 in the presence or absence of TAPI for 6 h at 37 °C. Cell supernatant levels of soluble TNF-RI and TNF-RII, as indicated, were quantified by ELISA. Shown is the mean (± S.D.) of at least three independent experiments. *, p < 0.05 versus untreated. B, neutrophils were either untreated or treated with the anti-Fas antibody CH-11 in the presence or absence of TAPI for 6 h, followed by TNFα stimulation for 3 additional hours at 37 °C. Cell supernatant levels of IL-8 were quantified by ELISA. Results are expressed as mean (± S.D.) of at least three independent experiments. *, p < 0.01 versus TNFα stimulation without TAPI.