1. by Asim Khwaja, and Louise Tatton
Caspase-Mediated Proteolysis and Activation of Protein Kinase Cδ Plays a Central Role in Neutrophil Apoptosis
by Asim Khwaja, and Louise Tatton
Blood
Volume 94(1):
July 1, 1999
©1999 by American Society of Hematology

2. Effect of the PKC inhibitor, GF109203X, on neutrophil apoptosis.
Effect of the PKC inhibitor, GF109203X, on neutrophil apoptosis. (A) Neutrophils were incubated in the absence (control) or presence of GF109203X (1 μmol/L), G-CSF (50 ng/mL), or GM-CSF (10 ng/mL) and apoptosis quantified by annexin V binding/flow cytometry at 24 and 48 hours. Mean ± standard error (SE) of 11, 10, and 8 separate experiments for GF109203X, G-CSF, and GM-CSF, respectively. (B) Concentration response curve for effects of GF109203X on neutrophil survival. Cell death was measured at 24 hours by annexin V binding and flow cytometry. Mean ± SE of three separate experiments.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

3. PKC isoforms expressed in neutrophils and HL-60 cells.
PKC isoforms expressed in neutrophils and HL-60 cells. Cell lysates from unstimulated freshly isolated neutrophils (PMN) or HL60 cells (20 μg of total protein were loaded per lane) were immunoblotted with various antibodies specific for PKC isoforms. Isoforms that were probed for, but not detected, in either cell type were PKCγ, ɛ, θ. Neutrophil/HL60 expression of the ERK2 MAPkinase is shown for comparison. Lysates from one of two separate experiments analyzed are shown.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

4. Incubation of neutrophils ex vivo leads to the generation of a C-terminal proteolytic fragment of PKCδ.
Incubation of neutrophils ex vivo leads to the generation of a C-terminal proteolytic fragment of PKCδ. Lysates prepared from neutrophils aged for the indicated times. Cells (20 μg of total protein loaded per lane) were analyzed by immunoblotting using an antibody that recognizes the C-terminal end of PKCδ (A) or antibodies to the C-terminal portion of PKCs  and βII (B).
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

5. Separation of neutrophils using annexin V–coated magnetic microspheres shows that PKCδ proteolysis precedes any detectable features of apoptosis.
Separation of neutrophils using annexin V–coated magnetic microspheres shows that PKCδ proteolysis precedes any detectable features of apoptosis. (A) Neutrophils were incubated for 12 hours ex vivo and then separated by using annexin V–coated microspheres, which bind to cells with external exposure of membrane phosphatidylserine, an early marker of apoptosis. Subsequent staining with FITC-conjugated annexin V and flow cytometry shows negative cells in the upper panel and positively selected cells in the lower panel. (B) Lysates prepared from the two fractions were analyzed by immunoblotting with anti-PKCδ antibody. Equal amounts of protein were loaded: lane 1, annexin V negative fraction; lane 2, annexin V positive. This experiment was performed twice with similar results.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

6. Measurement of PKC kinase activity in aged neutrophils.
Measurement of PKC kinase activity in aged neutrophils. (A) Neutrophils were incubated in the absence (t8h) or presence of the caspase-8 inhibitor, IETD.fmk (25 μmol/L), agonistic anti-Fas antibody (500 ng/mL), PKC inhibitor, GF109203X (1 μmol/L), or PKC inhibitor, Gö6976(1 μmol/L) for 8 hours. PKCδ was then immunoprecipitated using an anti–C-terminal antibody from neutrophil lysates and subjected to a kinase assay using histone H1 as a substrate. Due to the competitive, reversible nature of the PKC inhibitors (which would wash out during the immunoprecipitation protocol), further compound was added to a final concentration of 500 nmol/L to the kinase mixture before the addition of ATP. Phosphorylation of substrate was quantified after autoradiography and normalized to the time 0 value (100%). Results are mean ± SE of three separate experiments. (B) Representative autoradiograph from a PKCδ kinase assay. (C) PKCs  and βΙΙ were immunoprecipitated using anti–C-terminal antibodies after neutrophil aging for 8 hours in the absence or presence of the PKC inhibitor, Gö6976, and subjected to a kinase assay using histone H1 as a substrate as detailed above. Mean ± SE of three separate experiments.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

7. Effects of an agonistic anti-Fas/CD95 antibody on PKCδ proteolysis and neutrophil apoptosis.
Effects of an agonistic anti-Fas/CD95 antibody on PKCδ proteolysis and neutrophil apoptosis. (A) Neutrophils were incubated in the absence (control) or presence of agonistic Fas/CD95 antibody (500 ng/mL), PKC inhibitor, GF109203X (1 μmol/L), and caspase-8 inhibitor, IETD.fmk (25 μmol/L) and cell survival measured by annexin V binding using a flow cytometric assay. Mean ± SE of three separate experiments. (B) Neutrophils were incubated with agonistic Fas/CD95 antibody (500 ng/mL) with or without preincubation with the caspase-8 inhibitor, IETD.fmk (25 μmol/L) for the indicated times. Lysates were prepared, equal amounts of protein (20 μg) loaded per lane, and immunoblotted with an anti–C-terminal PKCδ antibody. (C) Neutrophils were incubated with agonistic Fas/CD95 antibody (500 ng/mL) for the indicated times. Lysates were prepared, equal amounts of protein (20 μg) loaded per lane, and immunoblotted with anti–C-terminal antibodies to PKC or PKCβ.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

8. Role of caspases in PKCδ proteolysis and neutrophil survival.
Role of caspases in PKCδ proteolysis and neutrophil survival. (A) Effect of caspase inhibitors on PKCδ proteolysis in neutrophils. Neutrophils were either lysed immediately after preparation (t0 con) or after 8 hours incubation in the absence (cont) or presence of broad spectrum caspase inhibitor, BD.fmk, the caspase-8 inhibitor, IETD.fmk (both at 25 μmol/L), the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L), or with agonistic Fas antibody (500 ng/mL) as indicated and immunoblotted with anti-PKCδ antibody. (B) Effect of recombinant caspases on PKCδ proteolysis. Purified recombinant caspase-3 or caspase-8 was incubated with recombinant PKCδ ± caspase inhibitor DEVD-CHO and proteolysis estimated by immunoblotting with an anti-PKCδ antibody. For comparison, a lysate from 8-hour aged neutrophils is shown (PMN). (C) Effect of caspase inhibitors on neutrophil survival. Neutrophils were incubated in the absence (cont) or presence of the broad spectrum caspase inhibitor, BD.fmk, the caspase-8 inhibitor, IETD.fmk (both at 25 μmol/L), or the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L) for 20 hours and survival measured by annexin V binding and flow cytometry. Mean ± SE of four separate experiments. (D) Effect of the caspase-8 inhibitor, IETD.fmk, and the PKC inhibitor, GF109203X, on apoptosis induced by an agonistic Fas antibody. Neutrophils were preincubated with IETD.fmk (25 μmol/L) or GF109203X (1 μmol/L) for 15 minutes and then anti-Fas antibody added (500 ng/mL). Cell survival was measured by annexin V binding in a flow cytometric assay. Mean ± SE of three separate experiments.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology

9. The effect of various agents on neutrophil production of ROS
The effect of various agents on neutrophil production of ROS. Freshly isolated neutrophils were incubated in the absence (control) or presence of the caspase-8 inhibitor, IETD.fmk (25 μmol/L), the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L), the PKC inhibito...
The effect of various agents on neutrophil production of ROS. Freshly isolated neutrophils were incubated in the absence (control) or presence of the caspase-8 inhibitor, IETD.fmk (25 μmol/L), the caspase-2 inhibitor, VDVAD.fmk (50 μmol/L), the PKC inhibitor, GF109203X, or an agonistic Fas antibody (500 ng/mL). Relative production of ROS was measured by a flow cytometric assay (DCF-DA) at the indicated time points. Mean ± SE of three separate experiments for VDVAD.fmk and four separate experiments for the rest.
Asim Khwaja, and Louise Tatton Blood 1999;94:
©1999 by American Society of Hematology