Correspondence to: Ben B. Whitlock, D505, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel:303-398-1282 Fax:303-398-1381 E-mail:whitlockb{at}njc.org.
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Abstract |
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The role of integrins in leukocyte apoptosis is unclear, some studies suggest enhancement, others inhibition. We have found that ß2-integrin engagement on neutrophils can either inhibit or enhance apoptosis depending on the activation state of the integrin and the presence of proapoptotic stimuli. Both clustering and activation of Mß2 delays spontaneous, or unstimulated, apoptosis, maintains mitochondrial membrane potential, and prevents cytochrome c release. In contrast, in the presence of proapoptotic stimuli, such as Fas ligation, TNF
, or UV irradiation, ligation of active
Mß2 resulted in enhanced mitochondrial changes and apoptosis. Clustering of inactive integrins did not show this proapoptotic effect and continued to inhibit apoptosis. This discrepancy was attributed to differential signaling in response to integrin clustering versus activation. Clustered, inactive
Mß2 was capable of stimulating the kinases ERK and Akt. Activated
Mß2 stimulated Akt, but not ERK. When proapoptotic stimuli were combined with either
Mß2 clustering or activation, Akt activity was blocked, allowing integrin activation to enhance apoptosis. Clustered, inactive
Mß2 continued to inhibit stimulated apoptosis due to maintained ERK activity. Therefore, ß2-integrin engagement can both delay and enhance apoptosis in the same cell, suggesting that integrins can play a dual role in the apoptotic progression of leukocytes.
Key Words: apoptosis, beta2 integrin, mitochondria, cytochrome c, neutrophil
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Introduction |
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Neutrophils have a short lifespan both in vivo and in vitro, undergoing spontaneous apoptosis with up to 80% death by 1620 h of in vitro culture ( (TNF
)1 receptor ligation (
ß2-integrins are important molecules for neutrophil function playing roles in cell activation, homing, and phagocytosis. In constitutively adherent cells, integrins play a critical role in supporting growth and survival (reviewed in M integrin chain on neutrophils was shown to inhibit spontaneous and Fas-mediated apoptosis (
M integrin chain deficient mice was delayed in vitro relative to control mice (
-induced apoptosis (
The ß2-integrin family consists of three ß dimers:
Lß2 (LFA-1),
Mß2 (Mac-1), or
Xß2 (p150,95). ß2-integrins are capable of interacting with a diverse array of ligands, but exist predominantly in an inactive, low affinity state (
The integrin-mediated downstream signals responsible for survival in anchorage-dependent cells are similar or identical to those used by growth factor receptors. Two kinase pathways that have received considerable attention are the PI-3K/Akt and extracellular signal-regulated MAPK (ERK) pathways. Both Akt and ERK are serine/threonine kinases that are invoked by numerous growth and survival factors. It may be possible, however, to override these antiapoptotic stimuli by potential proapoptotic insults. PI-3K and Akt activity is dramatically inhibited in fibroblasts exposed to stress stimuli, an effect attributed to the generation of ceramide (
Our data demonstrate different cellular responses upon clustering of inactive ß2-integrins vs. activation of ß2-integrins on neutrophils. We show that either Mß2 clustering or activation is differentially capable of activating Akt and/or MAPK-driven signaling pathways that prevent mitochondrial changes and inhibit spontaneous apoptosis. However, ß2-integrin activation, but not clustering, can enhance apoptosis stimulated by Fas ligation, TNF
, or UV irradiation. This combination of stimuli (integrin activation and proapoptotic stimuli) shifts the balance in the cell from an antiapoptotic to a proapoptotic posture by eliminating both Akt activation and mitochondrial protection. In contrast,
Mß2 clustering, in the absence of activation, continues to inhibit apoptosis in the presence of proapoptotic stimuli due to the activation of MAPK-driven signals.
Therefore, we propose a novel model, whereby the state of integrin activation and the environment in which the cell resides results in different survival outcomes. Clustered Mß2, in the absence of integrin activation, is universally antiapoptotic either during spontaneous or stimulated apoptotic progression. Downstream signals from activated ß2-integrins are also potentially antiapoptotic, however, in the presence of death stimuli, these survival signals are abrogated leading to enhanced cell death.
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Materials and Methods |
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Antibodies and other Reagents
The following mAbs were obtained from commercial sources and were confirmed to bind to human PMN by flow cytometry. Anti-M 2LPM19c, anti-
X KB90, and anti-HLA-ABC W6/32 were obtained from DAKO. Anti-
M Mo1 was obtained from Coulter Immunotech. Anti-
L MEM25, anti-
M VIM12, and anti-ß2 CLB-LFA1/1 were obtained from Caltag. Anti-ß1 MAR4, anti-ß3 VI-PL2, and anti-CD45 HI30 were obtained from BD PharMingen. Unless specified otherwise, mAbs were used at a final concentration of 1 µg/ml. All antibodies were tested as dialyzed preparations to rule out nonspecific effects of commercial solutions. Formyl-methionine-leucine-phenylalanine (fMLP) was obtained from Sigma-Aldrich and used at a final concentration of 10-7 M. TNF
was obtained from R&D and was used at a final concentration of 1,000 U/ml. Anti-Fas IgM clone CH11 was obtained from Upstate and used at a concentration of 0.5 or 1 µg/ml to induce apoptosis. Inhibitors of MEK (PD98059, 2'-amino-3'methoxyflavone and UO126, 1,2-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) and PI-3K (wortmannin and LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) were obtained from Calbiochem.
rhICAM-1coated Bead Preparation
Sulfate beads (1 µm; Molecular Probes) were washed in PBS and incubated with 10 µg of soluble recombinant human intracellular adhesion molecule 1 (rhICAM-1) in 0.9% saline with 20 mM Tris-HCL, pH 7.4, for 30 min at room temperature. We used rhICAM from two different sources (R&D and Alexa) with identical results. Beads were sonicated for 5 min, and resuspended in HBSS with 10 mg/ml LPS-free human serum albumin (HSA) for 30 min at room temperature. Beads were washed in HBSS and then resuspended in RPMI plus 1% BSA. This was added to PMN to give an approximate final concentration of 1 µg/ml rhICAM-1. Beads coated with HSA alone were used as controls in some experiments without effect.
Neutrophil Isolation
Human neutrophils were purified from whole blood as described previously (
Assessment of Apoptosis in Suspension Neutrophils
After isolation neutrophils were suspended at 2 x 106 cells/ml in RPMI supplemented with 1% LPS-free BSA (Sigma-Aldrich, A-1933) and incubated in conical polypropylene tubes (Sarstedt). Neutrophils, with or without ß2-integrin reagents, were either allowed to spontaneously apoptose over 10 h or were exposed to anti-Fas or TNF for 4 h. Experiments carried out with PI-3K and MEK inhibitors used a 30-min preincubation before addition of stimuli. After the requisite incubation 2 x 105 cells were diluted and cytocentrifuged at 600 rpm for 3 min on to glass slides and stained with a modified Wright-Giemsa stain. Cells were counted as apoptotic if their nuclei lost segmentation and appeared round and darkly stained. At least 200 cells were counted per slide in a blinded fashion. The accuracy of morphological assessment was checked periodically by flow cytometric analysis of hypodiploid DNA in propidium iodide stained neutrophils. Apoptotic percentages obtained by these two methods agreed within 5%.
Assessment of Apoptosis in Adherent Neutrophils
5,000,000 cells were suspended in 1 ml Ca2+/Mg2+-free Krebs-Ringer phosphate buffer, pH 7.2, containing 0.2% dextrose (KRPD) plus 0.25% HSA and labeled with a 111-Indium (111In; New England Nuclear) labeling mix consisting of KRPD/HSA and 4 x 10-3 M tropolonate. After a 5 min incubation at room temperature, cells were washed with KRPD/HSA and resuspended at 6 x 104 cells/ml in RPMI plus 1% BSA. This labeling procedure had no discernible effect on apoptotic progression. Labeled neutrophils were added to culture tubes or plated onto 15-mm glass coverslips coated overnight with 2.5 mg/ml fibrinogen (Calbiochem). Triplicate samples for each condition were set up to determine the total amount of radioactivity present. After a 46 h incubation, cells were gently fixed with dropwise addition of 0.5% glutaraldehyde. Cells were fixed for 30 min at 37°C or overnight at 4°C. Nonadherent cells were removed, washed, and cytocentrifuged. Cells in suspension were treated identically. Cytospin preparations and coverslips (adherent cells) were stained with 2 µg/ml 4',6-diamidino-2-phenylindole (DAPI; Calbiochem) in 50% methanol/PBS for 20 min. The slides and coverslips were washed gently in PBS and coverslipped or mounted using o-phenylenediamine. Cells were examined for apoptotic morphology by fluorescence microscopy with UV excitation, with 200210 cells counted per slide. The overall percentage of apoptotic cells was determined by multiplying the number of normal and apoptotic cells by the percentage of nonadherent versus adherent cells measured as radioactivity present in replicate wells. These adjusted numbers were then added to determine an overall percentage of normal and apoptotic cells.
Determination of Integrin Clustering or Activation
For visualization of M clustering, PMN were incubated with the mAbs 2LPM19 or VIM12 at 4 or 37°C for 45 min as above, centrifuged at 1,000 g at 4°C, and resuspended in isotonic 2% paraformaldehyde/sucrose/PBS. After a 20 min fixation and washing with KRPD/HSA, cells were incubated with 1:200 Cy3-conjugated goat antimouse (Jackson ImmunoResearch) for 30 min on ice, washed, resuspended in 510 µl, mounted onto glass slides with Gel Mount (Biomeda), and coverslipped. Stained cells were examined with a 63x water lens on a Vanox-T Olympus microscope. For
M activation, stimulated PMN were incubated 1 h on ice with 1.5 µg biotinylated CBRM1/5 (biotinylation was carried out using a commercial kit (Pierce Chemical Co.) or biotinylated anti-CD45 (BD PharMingen). Cells were washed and counterstained with 1:50 phycoerythrin-labeled streptavidin. Cells were analyzed by flow cytometry using FACscalibur (Becton Dickinson). Total ß2-integrin levels were measured on the same cells using FITC-labeled anti-CD18 (Caltag).
Fab and F(ab')2 Antibody Preparation
Digestion of the anti-M clone 2LPM19c and the anti-HLA-ABC clone W6/32 was accomplished using kits according to manufacturers instructions (Pierce Chemical Co,). Fab fragments were produced at 37°C using an immobilized pepsin slurry. F(ab')2 fragments were produced at 37°C using immobilized Ficin columns. Optimal digestion times were 12 h for Fab and 15 h for F(ab')2 fragments. Fab and F(ab')2 fragments were purified with protein A and fractions collected, assayed for protein content by absorbence at 280 nm, pooled, and dialyzed extensively against PBS over a period of 2436 h. The integrity of the fragments was assessed by nonreducing SDS-PAGE and silver stain, as well as Western blotting with HRP-labeled anti-mouse antibody. Bands of
110 kD and 50-kD bands were seen corresponding to F(ab')2 and Fab fragments, respectively. Neutrophils were stained with antibody fragments to confirm binding, and in addition, 2LPM19c fragments were tested for their ability to block fMLP-induced adhesion to confirm functionality of these fragments. All 2LPM19c fragments used in this study were able to inhibit adhesion as well as whole antibody.
ERK Immunoprecipitation and Kinase Assay
Lysates from isolated PMN, incubated with the appropriate stimulus at 37°C, were assayed for ERK activity using a enzyme activity reagent kit (Upstate) with slight modification of the provided protocol. After stimulation, 7.5 x 106 cells were centrifuged at 4°C and lysed with ice cold RIPA buffer (supplemented with 15 µg/ml leupeptin and aprotinin, 1 mM PMSF and 0.2 mM sodium orthovanadate). Lysates were centrifuged at 12,000 rpm at 4°C for 10 min. Supernatants were transferred to protein ASepharose (Zymed) beads containing 1 µg of rabbit anti-ERK2 antibody (Santa Cruz) and incubated for 2 h at 4°C with rotation. After incubation, beads were washed twice with cold lysis buffer and once with assay buffer (Upstate). Beads were resuspended in 50 µl assay buffer containing myelin basic protein, inhibitor cocktail, 1 µCi [32P]ATP (Amersham Pharmacia) and Mg/ATP cocktail. Beads were incubated for 10 min at 30°C with agitation. A portion of the reaction mix was transferred to P81 phosphocellulose paper, washed three times with 0.75% phosphoric acid, and analyzed by liquid scintillation counting.
Akt Immunoprecipitation and Kinase Assay
As with the ERK assays, Akt activity was measured using a enzyme activity kit (Upstate) with some modification of the provided protocol. PMN were lysed as in ERK assays. Supernatants were immunoprecipitated with proteinG Sepharose (Zymed) and 1 µg rabbit anti-Akt antibody (Santa Cruz). A purified rabbit polyclonal antibody to the unrelated kinase Rsk1 (Santa Cruz) was used as a control for specific immunoprecipitation of both Akt and ERK activity. Beads were incubated for 2 h at 4°C with rotation. Beads were washed as above and resuspended in 40 µl assay buffer containing substrate peptide (RPRAATF), protein kinase A inhibitor, 1 µCi [32P]ATP (1 µCi; Amersham Pharamcia), and Mg/ATP cocktail. Samples were incubated for 10 min at 30°C with agitation, centrifuged, and supernatants transferred to 20 µl 40% TCA, mixed, and incubated for 5 min at room temperature. A portion of the reaction mix was transferred to P81 phosphocellulose paper, washed as above and analyzed by liquid scintillation counting. Optimal activation times for Akt and ERK were determined for all appropriate stimuli.
Staining of Mitochondria
After stimulation, PMN were incubated with 10 µM JC-1 (Molecular Probes) at 37°C for 10 min, centrifuged, resuspended in cold PBS, and analyzed by flow cytometry with an excitation wavelength of 480 nm and 585/42-nm emission filter. Decreases in mean fluorescence intensity (FL-2) correspond to a loss of fluorescent JC-1 J aggregates in the mitochondria that occurs upon decreased mitochondrial membrane potential (
Cytochrome c (CytC) Immunostaining
Human PMN were incubated with stimuli for 1 or 1.5 h and CytC staining was performed as described elsewhere (
Data Analysis
Averages and SD values were calculated from at least three experiments. Statistical analysis was carried out using the JMP statistical program (SAS Institute, Cary, NC). The Tukey-Kramer and Dunnett's parametrical tests were used for single and multiple comparisons, respectively. Parametric tests were determined to be appropriate based on apparent normal distribution and equal variances (O'Brein test) of apoptosis data.
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Results |
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Clustering or Activation of ß2-Integrins Can Inhibit Neutrophil Apoptosis
Clustering of integrins in cell membranes contributes to the activation of survival signals (Mß2 clustering on neutrophils by direct engagement could modulate apoptosis. The experiments were performed under defined conditions with cells incubated in suspension, thus minimizing surface interaction. In this circumstance, integrin engagement with antibodies to either the
M or ß2 subunits delayed the apoptosis of human neutrophils (Fig 1 A). Anti-
M antibodies (2LPM19c, VIM12, and Mo1), an anti-ß2 antibody, and an anti-
L antibody, all inhibited neutrophil apoptosis by 3060% at 10 h. In contrast, antibody to
x, a component of the ß2-integrin p150,95, was largely ineffective, as were antibodies to ß1 and ß3 integrin chains. Antibodies to the nonintegrin surface molecules HLA and CD45 were also ineffective. A F(ab')2 fragment of 2LPM19c (hereafter referred to as 19c) was very effective at inhibiting neutrophil apoptosis indicating lack of Fc
R involvement (Fig 1 B). A monovalent Fab fragment of the same antibody had no effect, demonstrating that the apoptosis inhibition seen with 19c was not due to blocking cellcell proapoptotic adhesive signals (19c Fab and F(ab')2 were equally efficient at inhibiting adhesion), but rather a direct result of bivalent antibody engagement and ß2-integrin clustering or cross-linking (Fig 1 B). ß2-integrinmediated survival was not limited to antibody engagement as incubation of neutrophils with rhICAM-1, a natural ligand for ß2-integrins, immobilized on 1 µm beads also suppressed apoptosis (Fig 2). This suppression was due to ICAM-
Mß2 interactions since blocking anti-
M Fab (itself noninhibitory) completely reversed this suppression (Fig 2). Like antibody-mediated inhibition, ICAM-induced inhibition also required multivalent binding and clustering since soluble rhICAM-1, which exists largely in monomeric form (
Mß2 clustering was still evident late in culture (20 h), suggesting the induction of long-term survival signals in neutrophils (Fig 1 C).
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Since ß2-integrins exist largely in an inactive state and do not exhibit high affinity for ligands unless activated (Mß2 clustering and activation on apoptosis. In contrast to 19c, which blocks
Mß2-mediated adhesion by interacting with the major ligand binding domain (I-domain) of
M (
Mß2-dependent adhesion (
M Fab, but not a control Fab, suggesting dependence on ligand binding by
Mß2 in these suspension cultures.
Before further elucidating the mechanism of apoptosis inhibition via clustered or activated integrins, it was important to confirm M clustering and/or activation in our culture system. Both 19c, as well as the activating antibody VIM12, were capable of punctate membrane clustering of
Mß2 during 37°C incubation with antibodies, whereas a ring-like, uniform distribution of
Mß2 was seen at 4°C (Fig 3 A). Incubation of cells at 37°C in the presence of rhICAM beads and Mn2+ also resulted in
Mß2 clustering (data not shown). To determine the activation state of
M, we used the activation epitope antibody CBRM 1/5 kindly supplied by Dr. T. Springer (Harvard Medical School, Boston, MA;
Mß2 in the presence of 19c could not be determined due to steric hindrance between this antibody and CBRM1/5, as seen by the complete abrogation of fMLP-stimulated upregulation of CBRM1/5 binding (Fig 3 D). However, 19c does not appear to activate
Mß2, insofar as it does not stimulate cell adhesion or aggregation (
Mß2. This apparent lack of activation was not due to a masking of
Mß2 activation, like that seen with 19c, since fMLP-stimulated neutrophils coincubated with rhICAM beads and CBRM1/5 showed unaffected upregulation of CBRM1/5 epitope expression (Fig 3 C). The lack of ß2-integrin activation by rhICAM is further supported by the observation that the binding of rhICAM beads did not increase significantly over time (up to 4 h) suggesting that the binding of rhICAM did not lead to any measurable increase in adhesion (data not shown). Overall, our data support that 19c and rhICAM beads elicit ß2-integrin clustering without leading to any substantial activation; whereas VIM12 and Mn2+ are capable of activating ß2-integrins leading to increased neutrophil adhesiveness.
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Differential Activation of PI-3 Kinase and MAPK (ERK) Pathways after Mß2 Clustering or Activation
Studies in adherent cells have implicated PI-3K and ERK pathways in mediating integrin survival signaling (Mß2 clustering with either 19c F(ab')2 or rhICAM-1 coated beads (Fig 4 A). This pattern of reversal was also seen with the MEK inhibitor UO126 and the PI-3K inhibitor LY294002 (data not shown) indicating likely specificity for MEK and PI-3K. This nearly complete reversal of integrin clustering-mediated survival suggested major involvement of PI-3K and MEK driven pathways in the inhibition of apoptosis. In contrast,
Mß2 activation-mediated inhibition with either VIM12 or Mn2+ was reversed by wortmannin or LY294002, but was unaffected by the MEK inhibitors (Fig 4; LY294002 and UO126, data not shown), indicating involvement of PI-3K pathways only. Thus, a clear difference between signals induced by ß2-integrin activation and clustering is suggested.
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The chemotactic peptide fMLP was included in these experiments because of its known, characterized, ability to activate ß2-integrins in neutrophils (Fig 3 B; Mß2-dependent mechanisms. However, fMLP activates via signaling from the fMLP receptor to the integrin (inside-out activation). Unlike outside-in activation with VIM12 and Mn2+, suppression by fMLP was reversed by both wortmannin and PD98059 (Fig 4 A). This is consistent with the known ability of fMLP to activate MEK driven pathways in neutrophils (
Mß2.
Due to the possible difference between the survival pathways elicited by the clustering of inactive integrins and integrin activation, we postulated differences in downstream kinase activation. Akt and ERK are serine/threonine kinases that are downstream of PI-3K and MEK1, respectively, and are known to be involved in survival signaling by maintaining an antiapoptotic balance among Bcl-2 family members (Mß2 by 19c (intact antibody or F(ab')2), or rhICAM beads, activated both Akt and ERK as measured by in vitro kinase assays with immunoprecipitated enzymes at 20 min of stimulation (Fig 4B and Fig C). As expected, the activation of Akt was inhibited by wortmannin and ERK by PD98059. Fab fragments of 19c were inactive (data not shown), thus correlating with their lack of effect on apoptosis. By contrast, VIM12 and Mn2+ stimulated Akt activity, without activating ERK (Fig 4B and Fig C). These observations, combined with the inhibitor experiments (Fig 4 A), provide good evidence for a difference in the antiapoptotic signaling generated via
Mß2 clustering versus activation.
While both Mß2 clustering and activation stimulated Akt activity, the kinetics was somewhat different. Clustering-induced Akt activity appeared at 10 min, peaked at 1520 min, and was undetectable by 30 min. After integrin activation, however, Akt activation continued to increase over the entire time course (30 min).
Mß2-mediated Protection Against Mitochondrial Damage
One of the hallmarks of apoptotic changes is the loss of mitochondrial membrane potential and release of CytC. Although neutrophils do not contain large numbers of mitochondria (Mß2 clustering and activation (Fig 5 B). This block in fluorescence decrease is shown graphically in Fig 5 C, where the data are shown as percent fluorescence increase over control cells at 4 h. Both 19c and VIM12 yield an
50% increase in JC-1 fluorescence over that of control (4 h) cells indicating a maintenance of mitochondrial membrane potential. In addition, we examined the presence of CytC in neutrophils via immunofluorescence to confirm a possible correlation with the loss of mitochondrial potential. As seen in the top of Fig 5 D, CytC could be detected in fresh neutrophils, and costaining with a mitochondrial dye (MitoTracker Red) revealed a cytoplasmic punctate pattern (localization that agrees well with JC-1- and MAB1273 staining; Fig 5 A). The amount of CytC detected after 1.5 h in culture was significantly decreased (Fig 5 D, bottom, Control), indicating a spontaneous release of CytC from the mitochondria. However, integrin clustering or activation with 19c or VIM12 prevented the apparent release of CytC. Combined, these data provide good evidence that mitochondrial protection is a downstream mechanism for integrin-mediated inhibition of spontaneous neutrophil apoptosis.
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The Antiapoptotic Effect of ß2-integrin Activation Is Switched to Apoptosis Enhancement and Mitochondrial Damage in the Presence of Death Stimuli
While the experiments described above show that Mß2 engagement, either by clustering, or by activation/ligand interaction, inhibit neutrophil apoptosis, several reports suggest that ß2-integrins may also be involved in enhancing apoptosis (
(
Mß2 engagement could have differential effects on survival, depending on the integrin activation state and the presence or absence of other stimuli. The data shown in Fig 6 A support this concept. The activating antibody, VIM12, as well as Mn2+ enhanced apoptosis induced by anti-Fas, TNF
, or UV irradiation. By contrast, clustering of
Mß2 with 19c or rhICAM-1 beads before the addition of anti-Fas, TNF
, or UV irradiation continued to suppress apoptosis.
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This observation suggested that we may see an enhancement of induced mitochondrial damage in the presence of integrin activation. Fig 6 B shows JC-1 fluorescence as measured in neutrophils incubated for 4 h with no stimulus or with anti-Fas in the presence and absence of either Mß2 clustering (19c) or activation (VIM12). Treatment with anti-Fas resulted in a decrease in JC-1 fluorescence compared with control neutrophils (Fig 6b and Fig c). The combination of anti-Fas plus VIM12-induced
Mß2 activation dramatically enhanced this decrease. In contrast,
Mß2 clustering with 19c showed no change in anti-Fas induced mitochondrial changes, even though 19c suppresses anti-Fasinduced apoptosis. TNF
and UV-induced loss of mitochondrial membrane potential was also increased by VIM12 (data not shown), correlating with the enhancement of TNF
and UV-induced apoptosis. In addition, mitochondrial CytC release was also enhanced in the combined presence of anti-Fas and VIM12. Fig 6 D shows CytC in control (1 h) and anti-Fas treated neutrophils. The CytC immunostaining correlates with JC-1 fluorescence and is decreased in the presence of anti-Fas. An even greater loss of CytC staining is seen with the anti-Fas/VIM12 combination with no change seen with the anti-Fas/19c combination.
The data contained in Fig 6 is indicative of a major difference between the functional outcome of either ß2-integrin clustering or ß2-integrin activation and strongly suggest that activated Mß2 can serve a dual role, preventing mitochondrial damage and cell death in one situation, but aiding proapoptotic stimuli by enhancing mitochondrial damage and cell death in another.
Proapoptotic Stimuli Abrogate Mß2-induced Akt Activation
The enhancement of stimulus-induced neutrophil apoptosis seen in the presence of Mß2 activation suggested that the integrin-activated survival signals might be disrupted. To investigate this, ß2-integrinmediated Akt activation, common to clustering and activation, was examined in the absence or presence of anti-Fas IgM (Fig 7). VIM12 and Mn2+ stimulated Akt activity was entirely inhibited in the presence of Fas ligation, as was 19c and rhICAM bead stimulated Akt activity. Therefore, the switch from integrin-mediated survival and mitochondrial protection to integrin-mediated death enhancement may result from an active downregulation of Akt survival mechanisms.
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Surprisingly, anti-Fas alone activated Akt to a comparable degree to that of Mß2 clustering or activation (Fig 7). It was the combination of these stimuli that lowered Akt activity to control levels. This strongly suggests that signals originating from Fas and
Mß2 can synergize to prevent the activation of Akt.
Mß2 Clustering Continues to Inhibit Stimulated Apoptosis in the Absence of Akt Activation Due to MAPK Activation
As noted above, both Mß2 clustering and
Mß2 activation can synergize with Fas to eliminate Akt activation. However, unlike ß2-integrin activation, clustering of ß2-integrins continued to inhibit stimulated apoptosis in the absence of mitochondrial protection (Fig 6) or Akt activity (Fig 7). As indicated in Fig 4, clustering of
Mß2 via 19c or rhICAM beads activated ERK in addition to Akt. Thus,
Mß2 clustering may still be capable of activating ERK in the presence of Fas ligation. We tested this hypothesis and found that ERK activity was unaffected by coincubation with anti-Fas (Fig 8 A). In conjunction with these experiments we found that the sustained inhibitory effect of 19c or rhICAM beads on anti-Fasinduced apoptosis could be reversed by the MEK inhibitor PD98059 (Fig 8 B). This observation suggests that ERK can compensate for the loss of PI-3Kdependent Akt activity in neutrophils with clustered, inactive
Mß2 and inhibit stimulated cell death in a manner that appears to be independent of mitochondrial protection (Fig 6b, Fig c, and Fig d).
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Adherence of Neutrophils to Fibrinogen Inhibits Apoptosis in a Manner Identical to ß2-integrin Clustering
All of the experiments to this point were carried out on neutrophils in suspension. The fact that ß2-integrins are the primary adhesion molecules on neutrophils and can mediate adhesion to a wide variety of ligands raised the likelihood that ß2-integrin mediated surface engagement would also modulate neutrophil apoptosis.
To clearly determine surface effects on apoptosis, a special adhesion system was established. The experiments were performed on fibrinogen-coated glass coverslips using a very low cell number (6 x 104 cells/ml compared with 2 x 106 cells/ml in suspension incubations) to maximize cell-surface interaction and minimize cellcell interaction. At more normal cell concentrations, and as in most studies of these phenomena (
Using these methods, neutrophils on fibrinogen were shown to exhibit decreased spontaneous apoptosis compared with those (at an equal cell concentration) incubated in suspension (Fig 9). As expected, the surface mediated-survival was reversed by wortmannin and PD98059 in a fashion identical to the Mß2 clustering agents 19c and rhICAM-1 beads. Since neutrophils interact with ligands like fibrinogen with high affinity upon activation, it was important to examine this phenomenon in the presence of fMLP. Like unstimulated neutrophils, fMLP-stimulated neutrophils exhibited significant increased survival on fibrinogen, an effect that was greater than unstimulated cells and was significantly reversed by wortmannin and PD98059 (Fig 9). This agrees with the inhibitory effects of fMLP in suspension and the ability of fMLP to directly activate MAP kinase pathways. The increase in inhibition seen in the presence of fMLP plus fibrinogen was also seen in combination with rhICAM beads (data not shown). Therefore, these data further support a role for ß2-integrin engagement in the inhibition of neutrophil apoptosis and suggest that the interaction of both inactive and active ß2-integrins with different surface bound natural ligands, in the absence of proapoptotic signals, are capable of significantly inhibiting apoptosis.
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Discussion |
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We conclude that a significant function for ß2-integrins on neutrophils is the modulation of cell survival. As outlined in the introduction, some studies have implicated integrins in either promoting neutrophil survival or enhancing apoptosis, depending on the type of integrin engagement and/or the presence of proinflammatory or proapoptotic stimuli. The current study explains these apparent contradictions by establishing a dual role for ß2-integrins in cell survival and provides a potential signaling explanation for this observation. This dual role is a result of a clear difference between the functional outcome and signals induced by ß2-integrin clustering versus activation (Fig 10).
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The engagement of inactive Mß2 with 19c, a mAb that interacts at or near the I-domain of
M (Fig 1), was capable of inhibiting the spontaneous apoptosis of human neutrophils via activation of ERK and Akt, followed by mitochondrial protection. We believe that this requires some degree of
Mß2 clustering since bivalent molecules (whole or F(ab')2) were required to elicit this effect. In addition, VIM12, an antibody capable of activating
Mß2 (
Like the mAb 19c, the natural Mß2 ligands ICAM and fibrinogen, when immobilized, were capable of inhibiting spontaneous neutrophil apoptosis (Fig 2 and Fig 9), a phenomenon that we attribute to integrin clustering. We can detect a small, consistent percentage of resting neutrophils bound to ICAM-1 beads and fibrinogen-coated coverslips in the absence of measurable CBRM1/5 binding (data not shown), suggesting that unactivated
Mß2 is capable of binding immobilized ligands to some degree. A low percentage of adhering cells is not surprising given the low affinity of unactivated
Mß2 for ligands like ICAM. However, a recent report indicates that low affinity
Lß2/ICAM interactions on resting T cells are capable of initiating clustering of inactive
Lß2 on the cell surface (
Mß2 and subsequent activation of Akt and ERK signaling pathways in the absence of a large degree of ligand binding. In addition, inhibition was observed, and antiapoptotic signaling pathways activated, in neutrophils stimulated with fMLP in the presence of immobilized ICAM or fibrinogen (Fig 9; ICAM data not shown). This observation is important because it indicates that irregardless of the state of
Mß2 activation, binding of natural ligands by ß2-integrins can inhibit spontaneous apoptosis to a high degree.
VIM12, Mn2+, and fMLP-mediated inhibition was blocked by anti-M Fab (Fig 2) indicating that ligand engagement by activated
Mß2 was necessary to inhibit apoptosis. Since these experiments were conducted in suspension cultures (minimal surface engagement), it is probable that this inhibition is a result of homotypic neutrophil aggregation (
Mß2 cross-linking. Therefore, while we distinguish between activation and clustering as two separate physical states of ß2-integrins (Fig 10), it is likely that activated
Mß2 are present as clusters in the membrane. Indeed, immunofluorescence confirms that VIM12 and Mn2+ clusters
Mß2 (Fig 3 A; Mn2+ data not shown). Unlike clustering of inactive
Mß2, apoptosis suppression mediated by activated
Mß2 was unaffected by MAPK (ERK) inhibition, but was reversed by PI-3K blockade (Fig 4 A). The activation of downstream effectors correlated with this, in that activation of Akt, but not ERK, was seen. It is interesting that activated
Mß2 is capable of inhibiting neutrophil apoptosis in the absence of ERK activation. This suggests that the activation of PI-3K pathways alone can, under certain circumstances, compensate for the loss of concomitant MAPK activation. It is possible that augmented signals from activated
Mß2 are capable of sustained activation of PI-3K pathways. As mentioned above, Akt remained active for a longer period upon activation of
Mß2 (still high at 30 min). This may allow for a greater degree of Akt-mediated mechanisms capable of maintaining mitochondrial membrane potential and inhibiting CytC release.
While Mß2 clustering and
Mß2 activation induces survival signals capable of inhibiting spontaneous apoptosis, differential effects were seen on apoptosis stimulated by anti-Fas, TNF
, or UV exposure. In this scenario, activated
Mß2 enhanced apoptosis, while inactive, but clustered,
Mß2 continued to inhibit apoptosis. A mechanistic basis for this switch from an antiapoptotic tone to a proapoptotic one is provided by the data showing that
Mß2 mediated Akt activation was abrogated in the presence of Fas ligation and TNF
(Fig 7; TNF
data not shown). The absence of this pathway potentially explains why activated
Mß2, which does not activate ERK, no longer inhibits in the presence of proapoptotic stimuli. In addition, the effect on mitochondrial membrane potential and CytC release further supports the notion that Akt may protect the neutrophil by stabilizing mitochondria; without this mechanism clustered, inactive
Mß2 no longer confers mitochondrial protection and activated
Mß2 enhances mitochondrial damage (Fig 6). It is important to note that Fas ligation alone activated some Akt (Fig 7). (The possibility that Fas can activate PI-3K or Akt is not unprecedented;
Mß2 can limit the activity of PI-3K stimulated pathways either directly, by not allowing activation and recruitment of molecules involved in PI-3K activation; or indirectly, by activating molecules that can inhibit the downstream signaling from PI-3K such as the inositol phosphatases SH2 domain-containing inositol 5-phosphate (SHIP) and PTEN (
Clustering of Mß2 with either 19c or ICAM-1 continued to inhibit stimulated apoptosis in the apparent absence of PI-3K/Akt mediated antiapoptotic signals (Fig 6 A). We attribute this to
Mß2 clustering-mediated activation of MAPK (MEK/ERK; Fig 4 and Fig 8). This suggests that a PI-3K/Akt-independent ERK pathway acts to inhibit anti-Fasinduced neutrophil apoptosis. This observation appears to disagree with a previous study from our laboratory (
Mß2 clustering is likely to be significantly longer than that seen with other stimuli and could allow for more antiapoptotic pathway activation. Blocking ERK activity with PD98059 did not cause
Mß2 clustering induced signals to become proapoptotic (Fig 8 B) as was the case with ERK-deficient
Mß2 activation (Fig 6 A). Activated
Mß2 (but not clustered, inactive
Mß2) leads to the production of oxidants that may directly damage mitochondria and therefore enhance apoptosis in the absence of ERK activity (B. Whitlock, unpublished observation). This is supported by the observation that apoptosis was significantly enhanced by TNF
on fibrinogen (data not shown), a scenario known to induce massive oxidant production (
Our data support an important role for Akt and ERK in neutrophils; however, downstream targets in these cells remain to be identified. Akt can modulate apoptosis through several potential targets, including the phosphorylation of proapoptotic family members such as Bad (B (
B inducible gene (
Unlike Akt, and with the exception of fMLP stimulation, ERK was only activated by integrin clustering, not activation. The fact that Mß2 clustering-mediated inhibition was completely reversed either by inhibition of PI-3K or MEK (Fig 4 A) suggests that both Akt and ERK must be activated and may have common targets, including those listed above. However, because ERK activity is responsible for the continued inhibition of stimulated apoptosis in the apparent absence of PI-3K/Akt activity and mitochondrial protection, it is likely that other MAPK targets exist downstream of Bcl-2-family regulation and CytC release. In support of this, a recent study found that B-Raf overexpression and constitutive ERK activation in fibroblasts prevented apoptosis via a mechanism independent of PI-3K/Akt, downstream of CytC release, and upstream of caspase 3 activation (
Our data raise the intriguing possibility that repetitive engagement of active or inactive ß2-integrins on circulating leukocytes as they pass through the microvasculature could contribute to their in vivo lifespan by delaying their intrinsic death program. We have earlier shown the need for cell deformation during microvascular transit (
On the other hand, integrin-mediated cellular survivals might be counterproductive in the more permanent adhesive environment of an inflammatory reaction, but here, as we show, the presence of TNF and other potentially proapoptotic stimuli (e.g., Fas ligand) would combine to reverse the protective effects of integrin signaling. Experiments in
M deficient mice support the notion of a likely role for ß2-integrins in the apoptotic progression of elicited inflammatory neutrophils (
Mß2, which could, in turn, stimulate apoptosis. Based on our data, however, we would propose an alternate interpretation, whereby extravasated neutrophils undergo apoptosis more quickly due to engagement of activated
Mß2 and a death signal (e.g., TNF
) at the inflammatory site. This enhanced death could provide, along with apoptotic cell uptake, an effective mechanism to resolve inflammation. Therefore, this dual role of integrins in leukocyte apoptosis suggests that ß2-integrins can influence survival throughout the life of the cell and, therefore, need to be considered in any discussion of the role of apoptosis in the control of leukocyte numbers and the resolution of immune responses.
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Footnotes |
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Ben B. Whitlock and Shyra Gardai contributed equally to the completion of this work.
1 Abbreviations used in this paper: CytC, cytochrome c; ERK, extracellular signal-regulated kinase; fMLP, formyl-methionine-leucine-phenylalanine; MAPK, mitogen-activated protein kinase; PI-3K, phopshatidylinositol-3 kinase; rhICAM-1, recombinant human intracellular adhesion molecule 1; TNF, tumor necrosis factor
.
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Acknowledgements |
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We thank P. McDonald, D. Richter, P. Hoffman, and A. de Cathelineau for technical advice, and S.C. Frasch and C.A. Ogden for advice and discussion.
This work was supported by the National Institutes of Health grants HL34303, GM48211, and HL60980. B.B. Whitlock is supported by a National Research Service Award (NRSA) training grant (HL07670-10).
Submitted: 28 June 2000
Revised: 6 October 2000
Accepted: 27 October 2000
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References |
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