ADAM17 activity and other mechanisms of soluble L-selectin production during death receptor-induced leukocyte apoptosis

Abstract

L-selectin is an adhesion molecule expressed by neutrophils that broadly directs their infiltration in to sites of inflammation. It is also present at relatively high levels in the serum of normal individuals. It is well established that L-selectin is efficiently shed from the surface of neutrophils upon their activation, a process that regulates its density and binding activity. Neutrophil programmed cell death is critical for the resolution of inflammation, and L-selectin downregulation is induced during this process as well. The mechanisms underpinning this latter process are much less understood, and were investigated in this study. Using a disintegrin and metalloprotease (ADAM)-17 radiation chimeric mice, we demonstrate for the first time that during early events of death receptor-mediated neutrophil apoptosis, L-selectin downregulation occurs primarily by ADAM17-mediated shedding. This was observed as well upon using shRNA to knock down ADAM17 expression in Jurkat cells, a well-studied cell line in terms of the molecular processes involved in the induction of apoptosis. These findings directly reveal that ADAM17 activity occurs during programmed cell death. Hence, the cleavage of particular ADAM17 substrates may be an additional component of the anti-inflammatory program initiated by apoptotic neutrophils. Of interest was that during later stages of induced leukocyte apoptosis, soluble L-selectin production occurred independent of ADAM17, as well as membrane events, such as blebbing and microparticle production. This process may provide an explanation for the lack of diminished serum L-selectin levels in ADAM17-null mice, and suggests a mechanism for the homeostatic maintenance of soluble L-selectin levels in the blood of healthy individuals.

Figure 1. L-selectin shedding occurs by murine neutrophils upon the induction of apoptosis

A, Mouse bone marrow-derived neutrophils (2×10⁶/ml) were either untreated or treated with TNFα, TNFα and cycloheximide (CHX), or TNFα, CHX, and TAPI. The cells were cultured at 37°C for 90 min prior to L-selectin detection and for 180 min prior to phosphatidylserine detection. Relative L-selectin surface expression levels and annexin-V reactivity were determined by flow cytometry, as described in the Materials and Methods. Data shown are representative of at least three independent experiments using neutrophils isolated from separate animals. Negative control antibody staining of untreated cells is indicated (Isotype). The x axis = Log 10 fluorescence. To assess soluble L-selectin levels, neutrophils (4×10⁶/ml) were treated as described above for 90 min at 37°C, and media supernatants from the respective samples were subjected to ELISA, as described in the Material and Methods. The ELISA data are the mean (± SD) of three independent experiments performed in duplicate.

Figure 2. ADAM17, but not ADAM8, mediates L-selectin shedding at early time points of induced apoptosis

A, Mouse bone marrow-derived neutrophils (2×10⁶/ml) from wild-type chimeras or ADAM17 chimeras were either untreated or treated with TNFα or TNFα and CHX for 90 min (panels marked L-selectin) or for 180 min (panels marked annexin-V) at 37°C. B, Mouse bone marrow-derived neutrophils (2×10⁶/ml) from ADAM8 knock-out mice were treated as described above. Relative L-selectin surface expression levels and annexin-V reactivity were determined by flow cytometry, and data shown are representative of at least three independent experiments using neutrophils isolated from separate animals. Negative control antibody staining of untreated cells is indicated (Isotype). The x axis = Log 10 fluorescence. Soluble L-selectin levels in the media supernatants were determined by ELISA, and data shown are the mean (± SD) of 3 independent experiments performed in duplicate.

Figure 3. shRNA specific for ADAM17 diminishes L-selectin shedding upon Fas engagement

A, Jurkat cells were transduced with a bicistronic lentiviral vector expressing GFP and shRNA directed against human ADAM17 or control shRNA, as described in the Materials and Methods. Transduction efficiency was >50%, as indicated by GFP expression. A representative dot plot indicating the proportion of GFP expressing cells is shown in panel 1. GFP-negative and -positive cells (left and right boxes, respectively, panel 1) were electronically gated and each population was analyzed for their relative expression levels of ADAM17 and ADAM10 by flow cytometry. Transduced Jurkats cells expressing ADAM17 shRNA were stained for cell surface ADAM17 (panel 2) or ADAM10 (panel 4). Transduced Jurkats cells expressing control shRNA were stained for ADAM17 expression (panel 3). B, The transduced Jurkats cells were treated with rhFasL in the presence or absence of TAPI, as indicated, at 37°C for 30 min. Relative L-selectin surface expression levels were determined by flow cytometry. Negative control antibody staining of all cells is indicated (Isotype). The x axis = Log 10 fluorescence. SSC = laser light side scatter. The data are representative of three independent experiments.

Figure 4. Soluble L-selectin is generated by Jurkat cells in the presence of TAPI at later time points of induced apoptosis

A, Jurkat cells (2×10⁶/ml) were incubated with the anti-Fas antibody CH-11 in the presence or absence of TAPI for the indicated time points at 37°C. The presence of soluble L-selectin in the cell supernatants was determined by ELISA. B, Jurkat cells (2×10⁶/ml) were incubated with the anti-Fas antibody CH-11 in the presence or absence of TAPI, or were left untreated, as indicated, for 4 hr at 37°C. Relative L-selectin or LFA-1 surface expression levels, and caspase activity (FLICA reactivity) were determined by flow cytometry. C, Jurkat cells (2×10⁶/ml) were incubated with the anti-Fas antibody CH-11 or were left untreated, as indicated, for 2 hr (left panel) or 4 hr (right panel) at 37°C. Relative ADAM17 surface expression levels were determined by flow cytometry. D, Jurkat cells (2×10⁶/ml) were incubated with the anti-Fas antibody CH-11 in the presence or absence of TAPI, as indicated (TAPI, CH-11 or CH-11, respectively) for 4 hr at 37°C. Tissue culture media supernatants from the two cell treatments were removed of cells by centrifugation, filtered (0.22µm), and then used to suspend previously untreated Jurkat cells (2×10⁶/ml). These cells were then incubated with PMA to induce their overt activation. Relative L-selectin surface expression levels were determined by flow cytometry. Negative control antibody (Isotype) staining of untreated cells is indicated (B–D). The x axis = Log 10 fluorescence. E, Jurkat cells were incubated in the absence or presence of the anti-Fas antibody CH-11 plus TAPI, as indicated, for 4 hr at 37°C. The tissue culture media supernatant from both cell treatments was divided into two equal aliquots, and one of the aliquots was subjected to filtration (0.22µm) then ultracentrifugation (100,000 × g) for 1 hr. The levels of soluble L-selectin in the two aliquots for both cell treatments were determined by ELISA. For panels B-D, data are representative of at least three independent experiments. Data in panels A and E are the mean (± SD) of 3 independent experiments performed in duplicate. *, p < 0.001 vs. untreated; **, p < 0.05 vs. untreated. Statistical significance was determined by an unpaired student t test.

Figure 5. ADAM17-deficient neutrophils release L-selectin at later time points of induced apoptosis

A, Mouse bone marrow-derived neutrophils (2×10⁶/ml) were either untreated or treated with TNFα or TNFα/CHX in the presence or absence of TAPI for 180 min at 37°C. B, Mouse bone marrow-derived neutrophils (2×10⁶/ml) from ADAM17 chimeras were either untreated or treated with TNFα or TNFα/CHX for 180 min at 37°C. For panels A and B, relative L-selectin or LFA-1 surface expression levels and annexin-V reactivity were determined by flow cytometry. Negative control antibody staining of untreated cells is indicated (Isotype). The x axis = Log 10 fluorescence. Data are representative of at least three independent experiments using neutrophils isolated from separate animals. C, Mouse bone marrow-derived neutrophils (2×10⁶/ml) from ADAM17 chimeras were either untreated or treated with TNFα/CHX at 37°C for the indicated time points. The presence of soluble L-selectin in the cell supernatants was determined by ELISA. Data are represented as the mean (± SD) of 3 independent experiments performed in duplicate.