Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:863-870.)
© 2002 by Society for Leukocyte Biology

Fas activation opposes PMA-stimulated changes in the localization of PKC{delta}: a mechanism for reducing neutrophil adhesion to endothelial cells

Bill Hendey, Chun Liu Zhu and Stephanie Greenstein

Department of Pharmacology, Rush University, Chicago, Illinois

Correspondence: Bill Hendey, Department of Pharmacology, Rush University, 2242 W. Harrison, Rm. 264, Chicago, IL 60612. E-mail: bhendey{at}rush.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
We have shown previously that Fas activation results in a partial reduction of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophil adhesion to endothelial cells. The reduction in adhesion precedes early membrane markers of apoptosis and is not associated with any loss of membrane integrity. Rather, Fas activation reduces the PMA-stimulated expression and aggregation of ß2 integrins responsible for endothelial adhesion. A possible signaling mechanism for Fas effects on adhesion is the localization of protein kinase C {delta} (PKC{delta}). Western blot and immunofluorescence studies indicated that 1 h of Fas activation is required to reduce PMA-stimulated translocation of PKC{delta} to the membrane and adhesion. Rottlerin, a PKC{delta} inhibitor, also reduced PMA-induced PKC{delta} translocation and adhesion. In contrast, Gö6976, an inhibitor of conventional PKC isotypes, did not affect PMA-stimulated PKC{delta} translocation or reduce adhesion. There was no additive effect of Fas activation and rottlerin on reducing adhesion, suggesting that both agents were using a common pathway.

Key Words: apoptosis • phosphatidylserine • integrin ß2 receptor • phospholipids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 REFERENCES
 
Polymorphonuclear neutrophils (PMN) have a limited life span, circulating in the bloodstream for 4–10 h [1 ]. Circulating PMN can be activated and recruited to sites of infection or inflammation by bacterial peptides and/or chemokines. The activated PMN then adhere to the endothelium, extravasate through the vessel, and migrate to the site of infection or inflammation [2 , 3 ]. Once at the site of inflammation or infection, PMN carry out their effector functions including phagocytosis, granule release, and toxic metabolite production [4 ]. Although activated PMN contribute to the destruction of the invading organisms, they can also cause tissue damage [1 ] and have been implicated in the development or exacerbation of various crises including ischemia-reperfusion injury, acute respiratory distress syndrome, and sepsis [1 , 3 4 5 6 7 ].

Apoptosis or programmed cell death not only regulates PMN life span but may also contribute to the resolution of inflammation by regulating the removal of PMN from the inflammatory site [8 , 9 ]. An early event in apoptosis is the externalization of phosphatidylserine (PS) to the cell surface. This change in the plasma membrane composition targets PMN for phagocytosis by macrophages [9 10 11 ]. There is also evidence that apoptotic PMN lose cellular functions including endothelial cell adhesion, chemotaxis, phagocytosis, and respiratory burst [8 , 12 13 14 15 ]. However, the relevance of loss of function to inflammatory control is not clear because it is not known if the loss of function occurs before PS targets the PMN for removal.

Our investigation of PMN adhesion to endothelial cells following the stimulation of the Fas "death" receptor indicated that endothelial cell adhesion is reduced partially within 1 h of Fas activation. The partial reduction in adhesion precedes the externalization of PS and other early markers of apoptosis and is not associated with any loss of membrane integrity [16 ]. Fas activation reduced the phorbol 12-myristate 13 acetate (PMA)-stimulated expression and aggregation of ß2 integrins responsible for endothelial cell adhesion [16 ]. Thus, the initiation of apoptosis by Fas activation could impair the recruitment of circulating PMN before the PMN display PS and are targeted for removal [16 ]. The signaling responsible for Fas activation effects on adhesion is not known. The role of protein kinase C (PKC) in mediating the Fas effects on adhesion is investigated here, because PKC appears to play contrasting roles in the adhesion and apoptotic pathways.

Many compounds that stimulate PMN adhesion also activate PKC [17 18 19 ]. One measure of PKC activation is its translocation to the membrane or cytoskeleton cell fractions. Such a translocation could place PKC in proximity to the ß2 integrin receptor. A variety of PKC isotypes are found in human PMN [19 20 21 ]. These include the conventional PKC(s), {alpha}, ßI, ßII, which require calcium and lipids for activation; a single, nonconventional PKC, PKC{delta}, which does not require calcium; and an atypical PKC, PKC{zeta}, which is only activated by phospholipids [19 20 21 ]. PMA can activate the conventional PKC(s) and the nonconventional PKC isozymes, resulting in their translocation [19 ].

PKC{delta} has been most studied within the context of apoptosis. Stimulation of apoptosis via Fas or tumor necrosis factor {alpha} (TNF-{alpha}) results in the ceramide-mediated translocation of PKC{delta} to the cytoplasm within 1 h of stimulation [22 ]. In the cytosol, PKC{delta} is then cleaved by caspase 3, creating an active, catalytic fragment [23 ] that can be observed within 2 h of Fas activation [24 ]. The PKC{delta} catalytic fragment phosphorylates the phospholipid scramblase, resulting in PS externalization [24 ]. PKC{delta} inhibitors can block PS externalization [24 ] and can delay the onset of apoptosis [22 , 25 , 26 ].

Fas receptor activation and agonists that cause adhesion appear to have opposite effects on the localization of PKC{delta}. Although Fas activation causes the cytosolic localization of PKC{delta} [22 ], PMA and other agonists cause the membrane and cytoskeletal localization of PKC{delta} [19 ]. If PKC{delta} translocation is required for adhesion, then Fas activation could reduce adhesion by opposing the membrane localization of PKC{delta}. The loss of PKC{delta} translocation could preclude its interaction with the signaling complex necessary for ß2-mediated adhesion. In this study, it is demonstrated that Fas activation opposes the PMA-stimulated membrane localization of PKC{delta} and that the cytosolic translocation of PKC{delta} coincides with a partial reduction in adhesion. In addition, an inhibitor with specificity for PKC{delta} reduces the PMA-stimulated membrane association of PKC{delta} and adhesion. These results indicate that Fas activation reduces adhesion partially through its effects on the localization of PKC{delta}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 REFERENCES
 
Materials
Monoclonal antibody (mAb) Ch-11 was purchased from PanVera (Madison, WI). PKC{delta} C-20 and PKC{zeta} C-20 are affinity-purified antibodies raised against peptides mapping at the carboxy terminus of PKC{delta} and PKC{zeta}, respectively, and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat F(ab')2 anti-rabbit immunoglobulin G (IgG)-fluorescein isothiocyanate (FITC) and goat F(ab')2 anti-rabbit IgG-horseradish peroxidase (HRP) were purchased from Leinco Technologies (St. Louis, MO). PMA was purchased from Sigma Chemical Co. (St. Louis, MO). Vitronectin was purchased from Collaborative Biomedical Products (Bedford, MA). Fibronectin was purchased from Becton Dickinson (Bedford, MA). Rat pulmonary microvascular endothelial cells (RLEC) were a gift from W. Joseph Thompson, Ph.D., at the University of South Alabama (Mobile). Hanks’ balanced salt solution (HBSS) was purchased from Gibco-Life Technologies (Rockville, MD). Sterile, endotoxin-free phosphate-buffered saline (PBS) and HBSS with Mg2+, Ca2+, and glucose were purchased from Life Technologies (Grand Island, NY).

PMN isolation
Human whole blood was obtained from healthy volunteers via venipuncture. Blood was collected in sodium-heparin tubes, and PMN were isolated using a single-step gradient (Polymorphprep, Nycomed, Oslo, Norway) [27 ]. The final preparation consisted of >97% PMN. Care was taken to use sterile endotoxin-free buffers and plastic ware to avoid the possibility that endotoxin stimulation may mitigate the effects of Fas activation.

Western blot
PMN were treated ± 1 µg/ml Fas-activating antibody CH-11 for 1 h, then stimulated ± 5 nM PMA for the indicated time in HBSS. The PMN were pelleted, and HBSS was removed and resuspended in 1 ml buffer A containing 0.2 M Tris, pH 7.5, 5 mM ethylenediaminetetraacetate, 5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 0.1 M phenylmethylsulfonyl fluoride, 200 µM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), 10 µM leupeptin, and 10 µg/ml aprotinin. PMN were sonicated briefly on ice. The lysate centrifuged at 800 g at 4°C for 10 min to remove nuclei and any unlysed cells. The lysate was transferred to 13.5 ml ultracentrifuge tubes and was then centrifuged at 100,000 g at 4°C for 1 h. The supernatant or "cytosolic" fraction was collected. The pellet was resuspended in 500 µl buffer B (buffer A containing 0.5% Triton X-100). The samples were incubated ice for 20 min and then sonicated before centrifugation as above. The "triton soluble" supernatant or "membrane" fraction was collected. The amount of protein in the samples was quantitated using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Samples, equalized for protein content, were run on a 7.5% polyacrylamide sodium dodecyl sulfate (SDS)-reducing gel. Western blot was performed essentially as in Hendey et al. [28 ]. Briefly, the gel was transferred to nitrocellulose, blocked with blocking buffer (5% milk, 1% Tween 20 in PBS) for a minimum of 1 h, rinsed three times with PBS-1% Tween 20, and incubated with 1:1000 dilution of antibody PKC{delta} C-20 for 1 h. Then followed three rinses with PBS-1% Tween 20 and incubation with a 1:15,000 dilution of goat F(ab')2 anti-rabbit HRP for 1 h, following three rinses with PBS-1% Tween 20. A chemiluminescence kit (Kirkegaard and Perry Inc., Gaithersburg, MD) was used to visualize the protein/antibody complex. Chemiluminescence was recorded on film and later digitized. An optical density step tablet (Eastman Kodak, Rochester, NY) was also digitized with each film to allow for calibration. The optical density of each treatment was analyzed on a Macintosh computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Adhesion assay
The adhesion assay was performed essentially as described in Greenstein et al. [16 ]. PMN were fluorescently labeled using the acetyloxy methyl ester of calcein, calcein-AM (Molecular Probes, Eugene, OR), as described [29 ]. After 1 h, the labeled PMN were centrifuged and washed twice with HBSS. The PMN were then incubated for 1 h at 37°C. Some of the PMN were treated with anti-Fas antibody CH-11 for the indicated time during the 1-h incubation period. In the case of the PKC inhibitors, the cells were treated with the inhibitors for the final 15 min during the incubation period. Following the incubation period, the cells were plated on endothelial cells.

Falcon 96-well plates coated with human fibronectin (Becton Dickinson Labware, Bedford, MA) were used for seeding RLEC at a density of 5 x 103 cells/well. RLEC were grown to confluence in high-glucose Dulbecco’s modified Eagle’s medium, 10% fetal calf serum, 50 U/ml penicillin G, and 50 µg/ml streptomycin sulfate. Microscopy was used to verify confluence. Eight wells were used for each experimental condition. The media was removed from the wells, and 100 µl calcine-labeled PMN were added for a final concentration of 4 x 105 PMN/well. PMA (5 nM) or buffer was added to the wells for a final volume of 200 µl/well. The PMN were allowed to attach for 30 min at 37°C. An initial fluorescence reading was done to assess baseline fluorescence. The unbound PMN were rinsed 2x in HBSS before returning the volume to 200 µl. A second fluorescence reading was taken to measure the residual fluorescence.

The percent adhesion for each condition of the experiment was calculated by dividing the mean residual fluorescence by the mean baseline fluorescence for each condition. Calculating the percent adhesion for each experiment allowed for the comparison of replicate experiments. Each experiment was repeated on different days using fresh preparations of PMN. The data from replicate experiments were averaged to determine the mean percent adhesion and the SE for each condition. The actual number of replicates (n) for each set of experiments is indicated in Results and the figure legends.

Immunofluorescence
Freshly isolated PMN (1x107) were diluted to 4 ml in HBSS and incubated for 1 h at 37°C. During the 1-h incubation, some of the PMN were exposed to the Fas-activating antibody CH11 for the entire hour, and others were not exposed to the antibody (0 time) or were exposed for the final 20 or 30 min of the incubation period. During the incubation period, an eight-chambered slide (Nunc, Rochester, NY) was coated with Vitronectin (10 µg/ml) for 40 min. Following incubation, 5 x 105 PMN were plated on the slide with added HBSS to a final volume of 200 µl. The dish was covered, and cells were allowed to attach at 37°C for 5 min. The PMN were then treated ± 5 nM PMA for 10 min. The cells were fixed in 4% paraformaldehyde in PBS for 4 min at room temperature and were permeablized with 0.25% saponin in PBS for 5 min. The cells were then incubated with blocking buffer (PBS with 2 µg/ml bovine serum albumin, 10% fetal bovine serum, 1 mM MgCl, 0.5 mg/ml NaN3, and the protease inhibitors, 200 µM AEBSF, 10 µM leupeptin, and 10 µg/ml aprotinin) for 30 min as described [30 ]. Fluid was removed, and the cells were incubated with 1 µg/ml PKC{delta} c-20 for 1 h. Cells were rinsed gently with blocking buffer three times and then treated with 1 µg/ml goat F(ab')2 anti-rabbit IgG-FITC secondary antibody for 1 h in the dark. Following secondary antibody treatment, PMN were rinsed 3x with blocking buffer, and the wells were filled with 200 µl blocking buffer to cover the bottom of the well completely. Cells were visualized via fluorescent microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
REFERENCES
 
Fas effects on PKC{delta} localization
Freshly isolated PMN were pretreated for 1 h ± Fas-activating mAb CH11 and then stimulated ± 5 nM PMA. Following lysis and fractionation, Western blots were performed as described in Materials and Methods. The PMA-induced increase in PKC{delta} in the triton-soluble (membrane) fraction was measured following 10 min of PMA stimulation (Fig 1A ). A time course of PMA effects on PKC{delta} indicated that the membrane association of PKC{delta} was highest at early time points of 5 (Fig. 1C) and 10 min (Fig. 1A) and decreased with time after PMA stimulation (unpublished results). The effect of Fas activation on the atypical PKC{zeta} was also examined. PKC{zeta} is insensitive to PMA [31 , 32 ], and neither PMA nor Fas activation had any effect on its localization (Fig. 1B) . The PKC{zeta} blot was stripped and reprobed with a PKC{delta} antibody (Fig. 1C) . The comparison of PKC{zeta} and PKC{delta} probes indicates that the effects of PMA and Fas on PKC{delta} are not likely a result of any artifacts of gel-loading and others. Thus, PKC{delta} localization is sensitive to PMA and Fas activation (Fig. 1B) , and the localization of PKC{zeta} appears independent of PMA and Fas activation.



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  		Figure 1. Effect of Fas activation and PMA on PKC localization. PMN were treated ± the Fas-activating antibody CH11 for 1 h and were then stimulated ± 5 nM PMA for 10 min (A) or 5 min (B, C). The triton-soluble membrane fraction was isolated, and equal protein was applied to SDS-polyacrylamide gel electrophoresis for separation and was transferred to nitrocellulose for Western blotting as described in Materials and Methods. (A) A PKC{delta} standard (Std.) was run to verify the reactivity of the antibody and the location of PKC{delta} on the gel. PMA stimulation induced an increase in the membrane association of PKC{delta}. In contrast, Fas activation reduced the PMA-stimulated increase in PKC{delta}. (B) In a repeated experiment, a Western blot was probed with a PKC{zeta} antibody. Neither PMA nor Fas activation had any impact on the localization of PKC{zeta}. (C) The Western blot from (B) was stripped and reprobed with the antibody to PKC{delta}. As in (A), PMA induces an increase in PKC{delta}, and Fas activation reduces the PMA-stimulated increase in PKC{delta}.

 
Figure 2 shows a quantitation of the Western blots. Treatment with 5 nM PMA for 5 min increased PKC{delta} translocation significantly (Fig. 2) . One hour of Fas activation alone caused a small increase in PKC{delta} translocation, but it did not differ statistically from control (Fig. 2) . More importantly, Fas activation significantly reduced PMA-induced membrane translocation of PKC{delta} to near control levels [Fig. 2 ; P>.05 using analysis of variance (ANOVA) with individual mean comparisons using Scheffe F-test; n=5].



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  		Figure 2. Quantitation of Fas and PMA effects on PKC{delta} localization. PMN were treated as in Figure 1 , and Western blots from five experiments were examined. The optical density of the PKC{delta} for each treatment was assessed as described in Materials and Methods. *, PMA stimulation induced an increase in PKC{delta} that was greater than all other treatments. Fas reduced the PMA-stimulated increase significantly in PKC{delta}. Fas activation alone and the combined Fas and PMA treatments were slightly greater than the no stimulation control condition, but these differences were not statistically significant (*, P<.05 using ANOVA with multiple comparisons using a Scheffe F-test; n=5).

 
Time course of Fas effects on adhesion and PKC{delta}
Previous work indicated that 1 h of incubation with two different Fas-activating antibodies resulted in a partial reduction of subsequent PMA-stimulated adhesion to endothelial cells [16 ]. This loss of adhesion was a result of Fas activation and not an artifact of antibody stimulation because a 1-h incubation with isotype-control antibodies was without effect on adhesion or subsequent markers of apoptosis [16 ]. Figure 3 shows an early time course of Fas activation on PMN adhesion to endothelial cells. The PMN were incubated in buffer for 1 h at 37°C ± the addition of the Fas-activating antibody CH11. CH11 was present for the entire incubation period or for the final 30, 20, or 10 min of the incubation. The control "0" min condition was incubated in buffer for 1 h but was not exposed to CH11. The PMN were then plated on endothelial cells and stimulated ± 5 nM PMA before adhesion was assessed. Ten or 20 min of Fas activation had no impact on adhesion; 30 min of Fas activation caused a decrease in adhesion, but this decrease was not statistically significant. One hour of Fas activation was required to reduce adhesion to endothelial cells (P>.05 using ANOVA with individual mean comparisons using Scheffe F-test; n=5).



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  		Figure 3. Time course of Fas activation on PMN adhesion to endothelial cells. The PMN were incubated with Fas-activating antibody CH11 for 1 h in HBSS or with HBSS alone for 1 h and were then stimulated ± 5 nM PMA to induce adhesion to endothelial cells. Shorter times of Fas activation were accomplished by incubating the cells with HBSS initially and then adding the Fas-activating antibody for the last 10, 20, or 30 min of the 1-h incubation period. Endothelial cell adhesion was measured as described in Materials and Methods. Shown are the mean ± SE for five experiments. The zero time point refers to PMN that were not exposed to Fas activation during the 1-h incubation time. An examination of the zero time point indicates that basal adhesion is below 10% of the PMN and that stimulation with 5 nM PMA increases PMN adhesion to endothelial cells to nearly 70%. Fas activation alone did not increase adhesion significantly at any time point. Some reduction of PMN-stimulated adhesion was noted after 30 min of Fas activation, but the reduction in adhesion was not statistically significant. *, One hour of Fas activation was required to reduce PMA-stimulated adhesion (P<.05 using ANOVA with multiple comparisons using a Scheffe F-test).

 
The effect of the time of Fas activation on PKC{delta} translocation was measured using immunofluorescence microscopy. As in the adhesion time course, freshly isolated PMN were treated with buffer for 1 h at 37°C. During the incubation period, some of the cells were treated with the Fas-activating antibody CH11 for the entire hour or for the final 20 or 30 min of the 1-h incubation period. The PMN were then stimulated ± 5 nM PMA for 10 min and were then fixed and prepared for immunofluorescence as detailed in Materials and Methods. The buffer-only-treated "Control" PMN show a diffuse staining consistent with the cytoplasmic localization of PKC{delta} (Fig. 4 ). Stimulation of the buffer-treated PMN with 5 nM PMA results in a change in the pattern of fluorescence, suggesting a membrane and/or cytoskeletal association (Fig. 4) . Addition of the Fas-activating antibody CH11 for the final 20 min of the pretreatment incubation does not interfere with the PMA-induced changes in localization. CH11 treatment for 30 min caused a decrease in the effects of PMA, but a 1-h treatment with CH11 appears necessary to block the effects of PMA on the localization of PKC{delta}. Thus, the effects of Fas activation on reducing PKC{delta} membrane association parallel the loss of adhesion.



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  		Figure 4. Time course of Fas activation on PKC{delta} localization. The time course of Fas activation was done as described in Figure 3 . The PMN were plated for 15 min and then stimulated for 10 min ± 5 nM PMA. The cells were fixed, permeablized, and stained with an antibody to PKC{delta} and then counter-stained with a FITC-labeled secondary antibody as described in Materials and Methods. Unstimulated control cells show a diffuse pattern of PKC{delta} localization characteristic of cytoplasmic localization. PMA-stimulated cells show a punctate and ring pattern of staining characteristic of membrane and cytoskeletal localization. Twenty minutes of Fas activation does not block the PMA-induced changes in PKC{delta} localization. After 30 min of Fas activation, there was some reduction of the effects of PMA on PKC{delta} localization. After 1 h of Fas activation, the localization of PKC{delta} in PMA-stimulated cells appears more diffuse with some cells resembling the unstimulated control cells. All panels are to the same scale, and the bar shown in the first panel is equal to 10 µm.

 
Effect of PKC inhibitors on PKC{delta} localization and on adhesion
Treatment with 10 µM of the PKC{delta} inhibitor rottlerin for 15 min prior to PMA stimulation reduced the membrane localization of PKC{delta} (Fig. 5 ). Another PKC inhibitor, Gö6976, which does not inhibit PKC{delta} but does inhibit the conventional PKC isozymes with an IC50 of <10 nM [33 ], was used as a control. Unlike rottlerin, treatment with Gö6976 did not reduce PMA-stimulated membrane localization of PKC{delta} (Fig. 5) .



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  		Figure 5. Effect of PKC inhibitors on PKC{delta} localization. PMN were treated with 10 µM of the PKC{delta} inhibitor rottlerin or with 20 nM of the cPKC inhibitor Gö6976 for 15 min. The PMN were then stimulated ± 5 nM PMA for 5 min, lysed, fractionated, and prepared for Western blot as in Figure 1 . (A) A Western blot of the membrane fraction is shown that is representative of four replicate experiments. The optical density of the four blots was determined as described in Materials and Methods and is shown in (B). *, PMA induced an increase in PKC{delta} localization to the triton-soluble "membrane" fraction. Treatment with rottlerin significantly reduced the membrane localization of PKC{delta} caused by PMA to a level that was not significantly different from control. In contrast, the combination of Gö6976 + PMA did not reduce PMA-stimulated membrane localization and was greater than the control or the rottlerin + PMA treatment (*, P<.05 using ANOVA with multiple comparisons using a Scheffe F-test; n=4).

 
Figure 5B shows the quantitation of four Western blot experiments. PMA stimulation increased PKC{delta} membrane localization significantly when compared with the unstimulated control condition. Rottlerin treatment reduced the PMA-stimulated increase in PKC{delta} membrane localization and did not differ statistically from the control condition. In contrast, Gö6976 treatment did not reduce PMA-stimulated membrane localization of PKC{delta} and was statistically greater than the control condition (P<.05 using ANOVA with multiple comparisons using a Scheffe F-test; n=4).

Adhesion assays were performed comparing the effects of Fas activation and the PKC inhibitors on adhesion. The PMN were treated ± 1 µg/ml Fas activation antibody CH11 in HBSS for 1 h. Some of the PMN were treated with HBSS buffer for 45 min, and then 10 µM rottlerin or 20 nM Gö6976 was added for the final 15 min prior to adhesion. The cells were plated on endothelial cells and stimulated ± 5 nM PMA, and the adhesion assay was performed as in Materials and Methods. Fas activation and the PKC{delta} inhibitor rottlerin reduced PMA-stimulated adhesion to endothelial cells (Fig. 6 ; P<.05 using ANOVA with a Scheffe F-test for multiple comparisons; n=4 replicate experiments). Treatment with the conventional PKC inhibitor Gö6976 appeared to reduce adhesion by a small amount, but this reduction in adhesion was not statistically significant. Combining the Fas and rottlerin treatments did not increase the loss of adhesion (Fig. 5) . The lack of a Fas and rottlerin additive effect suggests that Fas activation and PKC{delta} inhibition use a common mechanism to reduce adhesion.



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  		Figure 6. Effect of PKC inhibitors on PMA-stimulated adhesion. PMN were treated with the Fas-activating antibody CH11 for 1 h or with 10 µM rottlerin or 20 nM Gö6976 for 15 min before beginning the adhesion assay as described in Materials and Methods. Shown are the mean ± SE for each treatment from four replicate experiments. PMA stimulation caused an increase in PMN adhesion that was reduced by prior Fas activation. Treatment with the PKC{delta} inhibitor rottlerin also reduced PMA-stimulated adhesion. There was no additive effect of combining the Fas and rottlerin treatments. Treatment with the cPKC inhibitor Gö6976 did not reduce adhesion significantly (*, P<.05 using ANOVA with multiple comparisons using a Scheffe F-test; n=4).

 
DISCUSSION
In previous work, we have shown that Fas activation reduces PMA-stimulated adhesion of PMN to endothelial cells [16 ]. The reduction of PMA-stimulated adhesion resulting from Fas activation is not a result of a loss of PMN viability or membrane integrity but rather is associated with a reduction in the expression and aggregation of the integrin ß2 receptor responsible for adhesion [16 ]. It should be noted that the reduction of adhesion occurs after only 1 h of Fas activation when few cells display "early" membrane markers of PMN apoptosis, including PS externalization and loss of CD16 from the cell surface. Increasing the time of Fas activation to 3 h does not increase the reduction of adhesion but does increase PS externalization and loss of CD16 from the cell surface [16 ]. These results indicate that the reduction in adhesion resulting from Fas activation precedes early markers of apoptosis and is likely a consequence of early signal-transduction events initiated by Fas activation. An examination of the signaling cascade initiated by Fas activation and by PMA stimulation suggests that PKC{delta} may be a common player in the adhesion and apoptotic cascades. Specifically, Fas activation has been shown to cause the cytosolic localization of PKC{delta} [22 ]. In contrast, PMA stimulation results in the membrane association of PKC{delta}. We propose that Fas activation could reduce subsequent PMA-stimulated adhesion by opposing the membrane localization of PKC{delta}. If this is the case, it needs to be demonstrated that Fas activation can oppose the membrane localization of PKC{delta} associated with PMA stimulation.

To determine if Fas activation opposes the PMA-stimulated membrane localization of PKC{delta}, the membrane localization of PKC{delta} was measured using Western blot. PMA stimulation causes a rapid translocation of PKC{delta} to the triton-soluble "membrane" fraction (Fig. 1A) . Densitometric quantitation of the PMA and Fas effects on PKC{delta} localization indicate that 5 min of PMA stimulation caused a significant increase in the membrane association of PKC{delta} of nearly fourfold (Fig. 2) . One hour of Fas activation reduced the subsequent PMA-induced membrane localization of PKC{delta} (Fig. 1A and 1C) to near control values (Fig. 2) . Control experiments indicate that neither Fas nor PMA treatments have any effect on the localization of another PKC isozyme, PKC{zeta} (Fig. 1B) . The lack of effect of PMA on PKC{zeta} was expected, because PKC{zeta} is not activated by PMA [31 , 32 , 34 ]. A reprobe of the PKC{zeta} blot with PKC{delta} (Fig. 1C) confirms the results shown in Figure 1A , indicating PMA caused the translocation of PKC{delta} and that prior Fas activation opposes the subsequent effects of PMA on PKC{delta} membrane association.

A time course of Fas effects on adhesion indicates that 1 h of Fas activation is required to reduce PMA-stimulated adhesion to endothelial cells (Fig. 3) . A similar time course of Fas effects on the translocation of PKC{delta} indicates that 1 h of Fas activation is also required to block the PMA-stimulated translocation of PKC{delta} (Fig. 4) . These results indicate that there is a temporal correlation between the Fas effects on adhesion and PKC{delta} localization.

Although PKC inhibitors have been shown to block PMN adhesion, previous generations of PKC inhibitors have not been specific for PKC isotypes. However, the isolation and expression of PKC isotypes have allowed the development and selection of agents with some isozyme specificity [34 ]. To determine if Fas activation was reducing adhesion through a PKC{delta}-mediated pathway, rottlerin, an isozyme-specific inhibitor for PKC{delta} was used. Rottlerin inhibits PKC{delta} with an IC50 of 6 µM compared with an IC50 of 30–42 µM for PKC {alpha}, ß, and {gamma} and an IC50 of 80–100 µM for PKC {varepsilon}, {eta}, and {zeta} [34 ]. Cellular studies have indicated that the addition of 10 µM rottlerin to PMN reduced PKC{delta} activity without impacting the activity of other PKC isozymes [24 ]. In addition, rottlerin also affects the agonist-induced translocation of PKC{delta} [26 , 35 ]. It was determined that treatment of PMN with 10 µM rottlerin reduced the PMA-stimulated membrane localization of PKC{delta} (Fig. 5) and reduced PMA-stimulated adhesion (Fig. 6) . The effect of rottlerin on adhesion was similar to the partial reduction of adhesion that results from Fas activation (Fig. 6) . The combination of Fas activation and rottlerin did not lead to any enhanced loss of adhesion (Fig. 6) , suggesting that both agents were operating through the same PKC{delta}-mediated mechanism.

It should be emphasized that Fas activation and rottlerin only reduced PMA-stimulated adhesion partially. It is possible that the residual adhesion is the result of incomplete inhibition of PKC{delta} localization or activity. However, this may be unlikely given that the combined effect of the two inhibitors would likely have reduced any residual PKC{delta} activity. Rather, it is likely that PMA stimulation also initiates other signaling pathways that account for the remaining adhesion.

Another inhibitor, Gö6976, with specificity for cPKC isozymes [33 ] but not PKC{delta}, was used as a control for the rottlerin treatment. Gö6976 treatment did not affect PKC{delta} translocation (Fig. 5) and caused a small but statistically nonsignificant reduction in adhesion (Fig. 6) . Although these data confirm that the loss of adhesion as a result of rottlerin treatment was not likely because of any nonspecific effect of rottlerin on cPKC isozymes, the results were surprising because others have demonstrated that PMA-stimulated adhesion can be inhibited with Gö6976 [18 ]. However, the lack of a significant effect of the Gö6976 effect on adhesion may be a result of the concentration of Gö6976. We treated the PMN with 20 nM Gö6976, a concentration in line with the published IC50 of <10 nM for the inhibition of purified PKC{alpha} and ßI [33 ]. In cellular studies, 20 nM Gö6976 has been shown to inhibit PKC{alpha} activity in PMN [24 , 26 ]. More importantly, treatment of PMN with 20 nM Gö6976 does not inhibit PKC{delta} activity [24 , 26 ] or its localization (Fig. 5) . Although the low concentration of Gö6976 was a conservative choice to minimize any unexpected effects on PKC{delta}, it is possible that higher concentrations are necessary to fully inhibit one of the cPKC isozymes, PKCßII. Studies showing inhibition of PKCßII activity in PMN have used a fivefold higher concentration of Gö6976 [25 ]. Similarly, studies of Gö6976 inhibiting PMA-induced adhesion have also used the higher, 100 nM concentration [18 ]. Thus, it is possible that PKCßII also contributes to PMA-stimulated adhesion.

In addition to the cPKCs and PKC{delta}, there has been a study demonstrating that the atypical isozyme, PKC{zeta}, also plays a role in PMN adhesion [18 ]. Neither PMA nor Fas activation changed the membrane localization of this isoform appreciably (Fig. 1B) . The lack of a PMA effect on PKC{zeta} was not unexpected because it is a member of the atypical PKC family that is not activated by PMA [31 , 32 , 34 ].

Although rottlerin treatment and Fas activation reduce PMA-stimulated membrane association of PKC{delta} and PMN adhesion, they have contrasting effects on later events in the apoptotic cascade. The differing effects of rottlerin and Fas activation are likely a result of their contrasting effects on cytosolic PKC activity. Fas activation results in the translocation of PKC{delta} to the cytosol [22 , 36 ] where it is cleaved by caspase 3, releasing an active kinase fragment that can be observed within 2–3 h after Fas stimulation [23 , 36 , 37 ]. This catalytic fragment phosphorylates phospholipid scramblase, which results in the appearance of the early apoptotic marker PS externalization [24 ]. Likewise, the expression of the PKC{delta} catalytic fragment alone is sufficient to induce apoptotic morphology [36 ]. Unlike Fas activation, which is responsible for increased PKC{delta} cytosolic activity, rottlerin inhibits the activity of the cytosolic PKC{delta} and its catalytic fragment [24 ]. Rottlerin treatment can inhibit PS externalization [24 ] and PMN apoptosis [26 ]. Thus, treatment with rottlerin can mimic the early Fas-induced, partial reduction of PMN adhesion to endothelial cells but antagonizes the latter effects of Fas on PS externalization and apoptosis.

Because Fas activation reduces PKC{delta} membrane localization and not PKC{delta} activity, it appears that the reduction of the membrane association of PKC{delta} is sufficient to reduce adhesion. This observation is consistent with recent work indicating that PKC specificity is controlled by localization [38 39 40 41 ]. How the membrane association of PKC{delta} affects endothelial cell adhesion is not known. The most direct linkage between PKC{delta} and adhesion would be PKC{delta} translocation to the membrane and the direct phosphorylation of the ß2 integrin (CD18) receptors responsible for PMN adhesion to endothelial cells. Although the phosphorylation of integrin ß2 following PMA stimulation has been observed [42 ], site-directed mutagenesis of the phosphorylated residues has not altered ligand binding in response to PMA [43 ]. Thus, it is unlikely that adhesion is regulated by the direct phosphorylation of the ß2 receptor. Rather, it is likely that PKC{delta} participates in a membrane-associated signaling complex that regulates the changes in receptor avidity associated with stimulated adhesion. Fas activation would then act to reduce adhesion by reducing the participation of PKC{delta} in the membrane-associated signaling complex, resulting in a reduction of ß2 avidity. Such a model is in line with previous observations that Fas activation reduces the PMA-stimulated aggregation of the ß2 integrin receptors [16 ].

The fact that Fas activation reduces PMN adhesion stands in contrast to TNF-{alpha} effects on PMN adhesion. Although TNF-{alpha} is more generally used to "activate" endothelium to bind unstimulated neutrophils, TNF-{alpha} stimulation of PMN has also been shown to stimulate PMN adhesion to unactivated endothelial cells [44 ]. Specifically, a 5- to 15-min stimulation of PMN with TNF-{alpha} is sufficient to stimulate >50% of the PMN to adhere to endothelial cells [44 ]. In contrast, Fas activation for a comparable time did not increase adhesion of PMN (Fig. 3) . Although the Fas and TNF-{alpha} receptors are members of the same family and share structural similarities [45 ], such disparate findings are not unexpected. TNF-{alpha} may have proinflammatory or apoptotic effects depending on the TNF receptor, TNFR1 or TNFR2, and the signaling system associated with the receptor at the time of stimulation [45 ].

It is not clear if TNF-{alpha}-induced adhesion involves membrane association of PKC{delta}. There is indirect evidence suggesting that TNF-{alpha} stimulation of PMN translocates PKC{delta} to the membrane. TNF-{alpha} stimulates the colocalization of PKC{delta} with the TNFR2 receptor, and PKC{delta} is responsible for the serine phosphorylation and apparent desensitization of TNFR2 [46 ]. The colocalization of PKC{delta} with the TNFR2 was evident in immunoprecipitation experiments done between 5 and 30 min after TNF-{alpha} stimulation [46 ]. These results suggest a prolonged membrane localization of PKC{delta} in PMN following TNF-{alpha} stimulation. However, other studies indicate that TNF-{alpha} does not induce the membrane localization of PKC{delta} in U937 and HL-60 leukemic cell lines [22 ]. Further studies are needed to determine if TNF-{alpha} stimulates the membrane localization of PKC{delta} in PMN and if PKC{delta} membrane localization is required for TNF-{alpha}-stimulated adhesion.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grant #AI40253 to B. H.


   FOOTNOTES
 
Current address of Stephanie Greenstein: Robert H. Lurie Comprehensive Cancer Center, Northwestern University, 303 E. Chicago Ave., Chicago, IL 60611.

Received August 25, 2001; revised December 20, 2001; accepted January 16, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 REFERENCES
 

  1. Smith, J. A. (1994) Neutrophils, host defense, and inflammation: a double edged sword J. Leukoc. Biol. 56,672-686[Abstract]
  2. Albelda, S. M., Buck, C. A. (1990) Integrins and other cell adhesion molecules FASEB J 4,2868-2880[Abstract]
  3. Mariscalco, M. M. (1993) Leukocytes and the inflammatory response Crit. Care Med. 21,S347-S348[Medline]
  4. Albelda, S. M., Smith, C. W., Ward, P. A. (1994) Adhesion molecules and inflammatory injury FASEB J 8,504-512[Abstract]
  5. Lush, C. W., Kvietys, P. R. (2000) Microvascular dysfunction in sepsis Microcirculation 7,83-101[Medline]
  6. Windsor, A. C. J., Mullen, P. G., Fowler, A. A., Sugerman, H. J. (1993) Role of the neutrophil in adult respiratory distress syndrome Br. J. Surg. 80,10-17[Medline]
  7. Shanley, T. P., Warner, R. L., Ward, P. A. (1995) The role of cytokines and adhesion molecules in the development of inflammatory injury Mol. Med. Today 4310,40-45
  8. Squier, M. K. T., Sehnert, A. J., Cohen, J. J. (1995) Apoptosis in leukocytes J. Leukoc. Biol. 57,2-10[Abstract]
  9. Savill, J. (1997) Apoptosis in resolution of inflammation J. Leukoc. Biol. 61,375-380[Abstract]
  10. Bruckenheimer, E. M., Schroit, A. J. (1996) Membrane phospholipid assymetry: host response to the externalization of phosphatidylserine J. Leukoc. Biol. 59,784-787[Abstract]
  11. Fadok, V. A., Savill, J. S., Haslett, C., Bratton, D. L., Doherty, D. E., Campbell, P. A., Henson, P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages J. Immunol. 148,2207-2216[Abstract]
  12. Dransfield, I., Buckle, A., Savill, J. S., McDowall, A., Haslett, C., Hogg, N. (1994) Neutrophil apoptosis is associated with a reduction in CD16 (Fc{gamma}RIII) expression J. Immunol. 153,1254-1263[Abstract]
  13. Dransfield, I. S., Stocks, S. C., Haslett, C. (1995) Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis Blood 85,3264-3273[Abstract/Free Full Text]
  14. Whyte, M. K., Meagher, L. C., MacDermot, J., Haslett, C. (1993) Impairment of function in aging neutrophils is associated with apoptosis J. Immunol. 150,5124-5134[Abstract]
  15. Tanji-Matsuba, K., van Eeden, S. F., Saito, Y., Okazawa, M., Klut, M., Hayashi, S., Hogg, J. (1998) Functional changes in aging polymorphonuclear leukocytes Circulation 97,91-98[Abstract/Free Full Text]
  16. Greenstein, S., Barnard, J., Zhou, K., Fong, M., Hendey, B. (2000) Fas activation reduces neutrophil adhesion to endothelial cells J. Leukoc. Biol. 68,715-722[Abstract/Free Full Text]
  17. Jones, S., Knaus, U., Bokoch, G., Brown, E. (1998) Two signaling mechanisms for activation of alpaM beta2 avidity in polymorphonuclear neutrophils J. Biol. Chem. 273,10556-10566[Abstract/Free Full Text]
  18. Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G., Butcher, E. C. (1998) Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis J. Biol. Chem. 273,30306-30315[Abstract/Free Full Text]
  19. Kent, J., Sergeant, S., Burns, D., McPhail, L. (1996) Identification and regulation of protein kinase C-delta in human neutrophils J. Immunol. 157,4641-4647[Abstract]
  20. Sergeant, S., McPhail, L. C. (1997) Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils J. Immunol. 159,2877-2885[Abstract]
  21. Tsao, L., Wang, J. (1997) Translocation of protein kinase C isoforms in rat neutrophils Biochem. Biophys. Res. Commun. 234,412-418[Medline]
  22. Sawai, H., Okazaki, T., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Kishi, S., Umehara, H., Domae, N. (1997) Ceramide-induced translocation of protein kinase C-delta and -epsilon to the cytosol: implications in apoptosis J. Biol. Chem. 272,2452-2458[Abstract/Free Full Text]
  23. Ghayur, T., Hugunin, M., Talanian, R. V., Ratnofsky, S., Quinlan, C., Emoto, Y., Pandey, P., Datta, R., Huang, Y., Kharbanda, S., Allen, H., Kamen, R., Wong, W., Kufe, D. (1996) Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis J. Exp. Med. 184,2399-2404[Abstract/Free Full Text]
  24. Frasch, S. C., Henson, P. M., Kailey, J. M., Richter, D. A., Janes, M. S., Fadok, V. A., Bratton, D. L. (2000) Regulation of phospholipid scramblase activity during apoptosis and cell activation by PKC{delta} J. Biol. Chem. 275,23065-23073[Abstract/Free Full Text]
  25. Khwaja, A., Tatton, L. (1999) Caspase-mediated proteolysis and activation of protein kinase C delta plays a central role in neutrophil apoptosis Blood 94,291-301[Abstract/Free Full Text]
  26. Pongracz, J., Webb, P., Wang, K., Deacon, E., Lunn, O., Lord, J. (1999) Spontaneous neutrophil apoptosis involves caspase 3-mediated activation of protein kinase C delta J. Biol. Chem. 274,37329-37334[Abstract/Free Full Text]
  27. Hendey, B., Klee, C. B., Maxfield, F. R. (1992) Inhibitors of calcineurin inhibit neutrophil chemokinesis on vitronectin Science 258,296-299[Abstract/Free Full Text]
  28. Hendey, B., Lawson, M., Marcantonio, E. E., Maxfield, F. R. (1996) Intracellular calcium and calcineurin regulate neutrophil motility through a receptor identified by antibodies to integrins alpha v beta 3 Blood 87,2038-2048[Abstract/Free Full Text]
  29. Everitt, E. A., Malik, A. B., Hendey, B. (1996) Fibronectin enhances the migration rate of human neutrophils in vitro J. Leukoc. Biol. 60,199-206[Abstract]
  30. Lawson, M. A., Maxfield, F. R. (1995) Ca2+ and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils Nature 377,75-79[Medline]
  31. Hug, H., Sarre, T. F. (1993) Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 291,329-343
  32. Nishizuka, Y. (1992) Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C Science 258,607-614[Abstract/Free Full Text]
  33. Martiny-Baron, G., Kazanietz, M. G., Mischak, H., Blumberg, P. M., Kochs, G., Hug, H., Marme, D., Schachtele, C. (1993) Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976 J. Biol. Chem. 268,9194-9197[Abstract/Free Full Text]
  34. Hoffman, J. (1997) The potential for isoenzyme-selective modualtions of protein kinase C FASEB J 11,649-669[Abstract]
  35. De Witt, B. J., Kaye, A. D., Ibrahim, I. N., Bivalacqua, T. J., D’Souza, F. M., Banister, R. E., Arif, A. S., Nossaman, B. D. (2001) Effects of PKC isozyme inhibitors on constrictor responses in the feline pulmonary vascular bed Am. J. Physiol. Lung Cell Mol. Physiol. 280,L50-L57[Abstract/Free Full Text]
  36. Mizuno, K., Noda, K., Araki, T., Imaoka, T., Kobayashi, Y., Akita, Y., Shimonaka, M., Kishi, S., Ohno, S. (1997) The proteolytic cleavage of protein kinase C isotypes, which generates kinase and regulatory fragments, correlates with Fas-mediated and 12-O-tetradecanoyl-phorbol-13-acetate-induced apoptosis Eur. J. Biochem. 250,7-18[Medline]
  37. Denning, M., Wang, Y., Nickoloff, B. J., Wrone-Smith, T. (1998) Protein kinase C{delta} is activated by caspase-dependent proteolysis during ultraviolet radiation-induced apoptosis of human keratinocytes J. Biol. Chem. 273,29995-30002[Abstract/Free Full Text]
  38. Dempsey, E. C., Newton, A. C., Mochly-Rosen, D., Fields, A. P., Reyland, M. E., Insel, P. A., Messing, R. O. (2000) Protein kinase C isozymes and the regulation of diverse cell responses Am. J. Physiol. Lung Cell Mol. Physiol. 279,L429-L438[Abstract/Free Full Text]
  39. Mochly-Rosen, D. (1995) Localization of protein kinases by anchoring proteins: a theme in signal transduction Science 268,247-251[Abstract/Free Full Text]
  40. Mochly-Rosen, D., Gordon, A. S. (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity FASEB J 12,35-42[Abstract/Free Full Text]
  41. Mochly-Rosen, D., Kauvar, L. M. (2000) Pharmacological regulation of network kinetics by protein kinase C localization Semin. Immunol. 12,55-61[Medline]
  42. Chatila, T. A., Geha, R. S., Arnaout, M. A. (1989) Constitutive and stimulus-induced phosphorylations of CD11/CD18 leukocyte adhesion molecules J. Cell Biol. 109,3435-3444[Abstract/Free Full Text]
  43. Hibbs, M. L., Jakes, S., Stacker, S. A., Wallace, R. W., Springer, T. A. (1994) The cytoplasmic domain of integrin LFA-1 beta subunit: sites required for binding to ICAM-1 and phorbyl ester-stimulated phosphorylation site J. Exp. Med. 174,1227-1238[Abstract/Free Full Text]
  44. Gamble, J. R., Harlan, J. M., Klebanoff, S. J., Vadas, M. A. (1985) Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor Proc. Natl. Acad. Sci. USA 82,8667-8671[Abstract/Free Full Text]
  45. Baud, V., Karin, M. (2001) Signal transduction by tumor necrosis factor and its relatives Trends Cell Biol 11,372-377[Medline]
  46. Kilpatrick, L. E., Song, Y. H., Rossi, M. W., Korchak, H. M. (2000) Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils Am. J. Physiol. Cell Physiol. 279,C2011-C2018[Abstract/Free Full Text]



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