Inhibition of Neutrophil Apoptosis by Type 1 IFN Depends on Cross-Talk Between Phosphoinositol 3-Kinase, Protein Kinase C-δ, and NF-κB Signaling Pathways

KeQing Wang, Dagmar Scheel-Toellner, See Heng Wong, Rachel Craddock, Jorge Caamano, Arne N. Akbar, Mike Salmon and Janet M. Lord
J Immunol July 15, 2003, 171 (2) 1035-1041; DOI: https://doi.org/10.4049/jimmunol.171.2.1035

Abstract

Neutrophils are abundant, short-lived leukocytes with a key role in the defense against rapidly dividing bacteria. They enter apoptosis spontaneously within 24–48 h of leaving the bone marrow. However, their life span can be extended during inflammatory responses by several proinflammatory cytokines. Inappropriate survival of neutrophils contributes to chronic inflammation and tissue damage associated with diseases such as rheumatoid arthritis. We have previously reported that type I IFNs can inhibit both cytokine deprivation and Fas-induced apoptosis in activated T cells. IFN-β locally produced by hyperplastic fibroblasts within the pannus tissue of patients with rheumatoid arthritis contributes to the inappropriately extended life span of infiltrating T cells. Type I IFNs are equally effective at delaying spontaneous apoptosis in human neutrophils. In the work presented here we investigated the signaling pathways involved in mediating this effect. The antiapoptotic actions of IFN-β were targeted at an early stage of neutrophil apoptosis, occurring upstream of mitochondrial permeability transition, and were phosphatidylinositol 3-kinase (PI3K) dependent, as they were blocked by the PI3K inhibitor LY294002. Analysis of signaling pathways downstream of PI3K revealed that the antiapoptotic effect of type 1 IFN was inhibited by rottlerin, SN50, and cycloheximide, indicating requirements for activation of protein kinase C-δ, NF-κB, and de novo protein synthesis, respectively. Moreover, EMSA was used to show that the activation of NF-κB occurred downstream of PI3K and protein kinase C-δ activation. We conclude that type I IFNs inhibit neutrophil apoptosis in a PI3K-dependent manner, which requires activation of protein kinase C-δ and induction of NF-κB-regulated genes.

Neutrophils are terminally differentiated cells with a short in vivo life span of ∼24–48 h, and effete neutrophils die spontaneously by apoptosis (1, 2). During an inflammatory response neutrophils are recruited to the inflamed site, and once the infection is cleared any residual neutrophils die by apoptosis, ensuring that neutrophils are removed by phagocytes and that the inflammation is resolved rapidly (3), minimizing the risk of loss of their toxic cell contents to the surrounding tissues. Although apoptosis is an intrinsic cell process, it can be regulated by external factors. For example, the neutrophil life span can be extended at sites of infection by cytokines such as GM-CSF (4) and inflammatory mediators, including complement C5a and LPS (5). Inappropriate survival of neutrophils and persistence at sites of inflammation are thought to contribute to the pathology of chronic inflammatory diseases such as rheumatoid arthritis (RA)4 (6). Identification of the factors influencing neutrophil survival and their mode of action will thus provide novel potential targets for therapy in chronic inflammation (2).

We have shown previously that T cells isolated from synovial fluid of patients with RA are a population of highly differentiated CD45RBdull T cells with a high susceptibility to apoptosis in vitro (7). However, they appear to be kept alive in vivo by type I IFN (8, 9) produced by fibroblast-like and macrophage-like synoviocytes in pannus tissue (8), which suppresses T cell apoptosis. More recently, we have shown that the influence of type 1 IFN (IFN-β) extends to neutrophils, which are a major cell population in the synovial fluid of patients with RA. The in vitro apoptosis of peripheral blood neutrophils was delayed up to 50% by IFN-β (10). In this study we have investigated the mechanism by which type I IFN delays neutrophil apoptosis, including the site of inhibition of the apoptotic program and the signaling pathways involved.

The type 1 IFN receptor is a heterodimer with no intrinsic kinase activity. Receptor ligation leads to activation of nonreceptor tyrosine kinases of the Janus family (Tyk-2 and Jak-1) and the recruitment of downstream signaling elements, including STAT proteins 1 and 3 (11) and insulin receptor substrate proteins, to the receptor β-chain (12). Insulin receptor substrate-1 has been shown to bind to the p85 subunit of phosphatidylinositol 3-kinase (PI3K), resulting in enzyme activation (12). PI3K has emerged as a key signal transducer for survival factor receptors, including growth factors, cytokines, and integrins (reviewed in Ref.13). We have shown that the inhibition of T cell and neutrophil apoptosis by IFN-β is PI3K dependent (10), but the signaling events downstream of PI3K mediating enhanced survival have not been determined. The lipid products of PI3K, predominantly phosphatidylinositol 3,4,5-trisphosphate, induce translocation of protein kinase B (PKB) to the plasma membrane, where it is phosphorylated and activated by PDK1, and this pathway has been proposed as a major mediator of survival signals downstream of PI3K (14). However, we and others have shown that PKB is not activated by type 1 IFN (10, 12), suggesting the existence of an alternative target for PI3K lipid products in IFN signaling. Protein kinase C (PKC) is among a group of protein kinases, the AGC kinases, that are regulated by PDK1 and the lipid products of PI3K (15) and may represent the target of PI3K in the type 1 IFN signaling pathway.

PKC is a lipid-dependent serine/threonine protein kinase consisting of 11 isoenzymes, several of which have been shown to play a role in the regulation of apoptosis (16). PKC-α has been implicated in the prevention of apoptosis, as it is able to phosphorylate Bcl-2 and enhance its antiapoptotic actions (17). PKC has also been proposed to regulate the NF-κB signaling pathway (18, 19), which is responsible for the induction of several antiapoptotic genes (20). In contrast, PKC-δ is activated by proteolytic cleavage during apoptosis, mediated by caspase 3 (21), and its target substrates include the nuclear lamina (22), DNA repair enzymes (23), and scramblase, an enzyme involved in effecting exposure of phosphatidylserine on the surface of apoptotic cells (24). We have already shown that type 1 IFN modulates the subcellular location of PKC-δ, inhibiting the nuclear translocation of this isoenzyme and its cleavage by caspase 3 (25). Recently, activation of PKC-δ and PI3K by TNF-α has been shown to result in the prevention of apoptosis in neutrophils (26), revealing an antiapoptotic role for PKC-δ. PKC-δ may therefore have dual functions with respect to cell survival dependent upon whether it is activated by lipid cofactors or by caspase 3-mediated proteolytic cleavage, and IFN-β may be able to regulate both these events.

In the study reported here we show that IFN-β-induced survival of neutrophils involved PI3K-dependent activation of NF-κB and activation of PKC-δ, and survival was blocked by inhibitors of these signaling pathways and by cycloheximide. These data thus suggest that PI3K, PKC-δ, and NF-κB are key downstream effectors in the induction of type 1 IFN-mediated neutrophil survival.

Materials and Methods

Isolation and culture of human peripheral blood neutrophils

Twenty to 100 ml of venous blood was taken from healthy volunteers, and neutrophils were isolated on Percoll density gradients as described previously (27). Neutrophil preparations contained >98% neutrophils, and contaminating cells were mainly eosinophils. Neutrophils were resuspended in RPMI 1640 medium (Life Technologies, Gaithersburg, MD), supplemented with 10% heat-inactivated FCS (Sera-Lab, Loughborough, U.K.) and containing 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO). Neutrophils were either used immediately as healthy control cells or were cultured in a humidified 5% CO2 atmosphere in the presence or the absence of recombinant human IFN-β (BioSource, Camarillo, CA) or human type 1 IFN purified from fibroblast tissue culture supernatant (Sigma-Aldrich). For studies involving the PI3K inhibitor LY294002 (Calbiochem, La Jolla, CA), the classical PKC inhibitor Go6976 (Calbiochem), the PKC-δ inhibitor rottlerin (Calbiochem), or inhibitors of the NF-κB pathway (SN50; Calbiochem), cells were incubated with the inhibitors at the concentrations shown for 30 min before the addition of IFN-β. To determine the requirement for de novo protein synthesis, cycloheximide (1 μg/ml) was added to cell cultures at the same time as IFN-β. Preliminary studies established that this concentration was sufficient to fully inhibit protein synthesis in neutrophils (data not shown).

Measurement of neutrophil apoptosis

To determine the effect of type 1 IFN on spontaneous neutrophil apoptosis in vitro cytospin preparations (3 min, 10 × g; Cytospin 2; Shandon, Pittsburgh, PA) were made of freshly isolated or neutrophils cultured for up to 20 h in medium alone or in the presence of a range of concentrations of IFN-α or IFN-β (0.8–800 IU/ml). Cytospins were then differentially stained using a commercial May-Grunwald Giemsa stain (Diff-Quick; Gamidor, Abingdon, Oxfordshire, U.K.) and assessed for apoptotic morphology. Morphological assessments were confirmed by measurement of annexin V binding using a commercial kit (R&D Systems, Minneapolis, MN) and flow cytometric analysis.

Caspase activity assays

Activation of caspases was measured by assessing cleavage of a tagged caspase 3 (DEVD-7-amino-4-methyl-coumarin (DEVD-AMC)) or caspase 9 (LEHD-AMC) substrate peptide and release of the fluorochrome AMC. Fluorescence was measured at excitation and emission wavelengths of 355 and 460 nm, respectively, using a fluorimeter (PerkinElmer/Cetus, Norwalk, CT).

Assessment of mitochondrial permeability transition

Mitochondrial permeability transition was detected using a method based upon the ability of intact mitochondria to take up and retain cationic lipophilic fluorescent dyes (28). Neutrophils were loaded with 10 μM JC-1 (Molecular Probes, Eugene, OR) for 30 min at 37°C or 40 nM DiOC6 (Molecular Probes) for 10 min at 37°C. Cells were then washed in PBS, resuspended in ice-cold PBS, and analyzed by flow cytometry or confocal microscopy. Aggregates of JC-1 are retained in intact mitochondria and fluoresce red, whereas mitochondria that have undergone a permeability transition release the JC-1 monomers into the cytoplasm, where they fluoresce green. DiOC6 is retained by cells with intact mitochondria, but is lost when permeability transition occurs.

Measurement of PKB and PKC activation

Activation of PKB was measured by determining the phosphorylation of PKB as previously described (10). Whole-cell extracts precipitated with 10% TCA were probed by Western blotting using a phospho-PKB (Ser473)-specific Ab (New England Biolabs, Beverly, MA). PKB activity was also measured enzymatically using a commercial kit (Upstate Biotechnology, Lake Placid, NY) to determine the incorporation of [γ-32P]ATP into a PKB peptide substrate by immunoprecipitated PKB, following the manufacturer’s instructions. Phosphotransferase assays were conducted for 10 min at 30°C, the reaction was stopped with 40% TCA, and data were expressed as disintegratins per minute per 107 cells.

Activation of PKC isoenzymes was assessed by determining translocation of PKC from the cytosol to the cell membrane fraction. Neutrophils were lysed as previously described (29), and cytosol and cell membrane fractions were isolated by differential centrifugation. The phorbol ester PMA was used as a positive marker for PKC activation. Translocation of PKC isoenzymes was determined by Western blotting with isoenzyme-specific Abs to PKC-α (Transduction Laboratories, Lexington, KY), PKC-β, PKC-δ, or PKC-ζ (Santa Cruz Biotechnology, Santa Cruz, CA). HRP-conjugated anti-rabbit IgG and anti-mouse IgG (Amersham Pharmacia Biotech, Arlington Heights, IL) were used as secondary Abs, and blots were developed by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech).

Detection of NF-κB activation

Activation of the NF-κB pathway was detected by EMSA for NF-κB. Neutrophils were cultured with 800 IU/ml IFN-β in the absence or the presence of pharmacological inhibitors, or 10 ng/ml LPS as a positive control, for 20 min. Neutrophils (1 × 107) were disrupted by nitrogen cavitation, and nuclei were isolated by Percoll density centrifugation exactly as previously described (30). Ten micrograms of nuclear extract was combined with 0.5 μg of poly(dI-dC) (Amersham Pharmacia Biotech) and binding buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl2, 17% glycerol, 1 mM DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 2 mM AEBSF). Ten nanograms of 32P end-labeled, double-stranded oligonucleotide of sequence 5′-AGTTGAGGGGACTTTCCCAGGC-3′ was incubated with the extract for 15 min at room temperature. The specificity of probe binding was confirmed by competition with a 100-fold excess of cold oligonucleotide. DNA-protein complexes were separated on 5.5% polyacrylamide gels at 4°C with 0.5× tris-borate-EDTA as a running buffer. Gels were dried, and binding of radiolabeled oligonucleotide was detected by autoradiography.

Results

Effects of IFN-α and IFN-β on spontaneous neutrophil apoptosis and function in vitro

Neutrophils isolated from peripheral blood and cultured at 37°C died by apoptosis, with ∼60% of cells displaying apoptotic morphology after 20 h in vitro (Fig. 1⇓a). Culture of neutrophils in serum-free medium in the presence of a range of concentrations of recombinant human IFN-α and IFN-β confirmed that IFN-β was able to delay neutrophil apoptosis (10) and that the survival effects were concentration dependent, with inhibition detected at concentrations as low as 8 IU/ml and a maximal effect seen at 800 IU/ml (Fig. 1⇓b). Type 1 IFN purified from cell culture supernatants was effective over the same concentration range (Fig. 1⇓b), indicating that the apoptosis-inhibiting effects of the recombinant proteins were not due to contamination with bacterial products such as LPS. Moreover, neutrophils treated with IFN-β maintained their function and were able to generate superoxide in response to fMLP, a function that was much reduced in neutrophils cultured in the absence of IFN-β (Fig. 1⇓c). IFN-β was thus able to delay neutrophil apoptosis and extend their functional life span, factors that will contribute to the role of neutrophils in mediating the nonspecific tissue damage associated with chronic inflammatory diseases such as RA.

FIGURE 1.
FIGURE 1.

Type 1 IFN extends neutrophil life span and function. a, Isolated peripheral blood neutrophils cultured for 20 h in the absence (control) or the presence of 800 IU/ml IFN-β were differentially stained to identify apoptotic cells by morphology. b, Neutrophils were cultured for 20 h in serum-free medium in the absence or the presence of recombinant human IFN-α (•) or IFN-β (▪) or IFN-β purified from fibroblast culture supernatant (▴) over the range of concentrations shown. Apoptosis was measured by differential staining of cells using a modified Giemsa stain and examination of nuclear morphology to identify apoptotic cells. c, Superoxide generation in response to fMLP was determined in neutrophils cultured for 20 h in the absence (▪) or the presence (▴) of 800 IU/ml IFN-β. Data are from a single experiment that is representative of three performed.

Effect of IFN-β treatment on caspase activity and mitochondrial membrane integrity

To determine the stage in the apoptotic program at which IFN-β was acting to delay apoptosis we measured the effects of the cytokine at two distinct stages: at a relatively late stage, represented by caspase 3 activation, and at the earlier stage of mitochondrial membrane permeability transition and caspase 9 activation. Fig. 2⇓a shows that caspase 3 enzyme activity was increased during neutrophil apoptosis and was significantly reduced by IFN-β treatment. IFN-β was therefore acting upstream of caspase 3 activation. Fig. 2⇓b shows that caspase 9 was also activated during neutrophil apoptosis and that IFN-β significantly inhibited the activation of this caspase at 9 h of incubation, with a less marked inhibition after 20 h. The latter indicates that type 1 IFN delays rather than completely blocks neutrophil apoptosis, and by 48 h the majority of neutrophils had activated caspase 3 and died by apoptosis (data not shown). To confirm that caspase 9 was activated as a consequence of a mitochondrial permeability transition, mitochondrial membrane integrity was assessed. In neutrophils cultured overnight in medium alone (control), red fluorescing JC-1 was lost from the majority of mitochondria and fluoresced green in the cell cytoplasm, revealing that permeability transition had occurred in the mitochondria of apoptotic neutrophils (Fig. 2⇓c). IFN-β treatment prevented the loss of mitochondrial membrane integrity (Fig. 2⇓c), and JC-1 was retained in the mitochondria in a majority of cells and emitted red fluorescence (Fig. 2⇓c), showing that apoptosis was delayed at the mitochondrial stage or upstream of this event.

FIGURE 2.
FIGURE 2.

IFN-β inhibits caspase activity and maintains mitochondrial integrity. Freshly isolated neutrophils (To) or neutrophils cultured for up to 20 h in the absence or the presence of 800 IU/ml IFN-β were assessed for caspase 3 (a) or caspase 9 (b) activity using an assay based on the cleavage of a fluorescent substrate for caspase 3 (DEVD-AMC) or caspase 9 (LEHD-AMC), respectively. Data are the mean ± SD of three separate experiments. ∗, p < 0.05; ∗∗, p < 0.01. c, Neutrophils that were cultured for 20 h in medium alone (control) or for 20 h in the presence of 800 IU/ml IFN-β were labeled with JC-1 for 10 min before analysis of fluorescence by confocal microscopy. JC-1 is retained by intact mitochondria and fluoresces red; it is lost by those that have undergone a permeability transition and fluoresces green in the cytoplasm. The image shown is representative of four similar experiments performed.

PI-3K dependence of IFN-β effects on neutrophil apoptosis

Fig. 3⇓a shows that the inhibition of neutrophil apoptosis by IFN-β was significantly reduced in the presence of LY294002, confirming our previous data (10) and that the effects of the PI3K inhibitor were concentration dependent and reached a maximal effect at 25 μM. This concentration of LY294002 has been shown to produce a near-maximal inhibition of PI3K activity (31). Moreover, apoptosis was assessed in this study by measuring mitochondrial permeability transition using the mitochondrial dye DiOC6 and FACS analysis. DiOC6 is retained by cells with intact mitochondrial membrane potential and is lost when permeability transition occurs; the DiOC6high cells therefore represent nonapoptotic neutrophils. The results indicate that IFN-β actions at the mitochondrial level are also mediated through PI3K activation. PKB has been proposed as a major effector of PI3K signaling for cell survival (14), but Fig. 3⇓, b and c, confirms that PKB was not activated by IFN-β in neutrophils (10, 12), indicating that an alternative downstream target was inhibiting apoptosis. The positive control of fMLP gave a rapid activation of PKB in both the anti-phospho-PKB Western blot assay (Fig. 3⇓b) and an in vitro kinase assay (Fig. 3⇓c).

FIGURE 3.
FIGURE 3.

The effects of IFN-β are PI3K dependent. a, Neutrophils were cultured for 20 h in medium containing 800 IU/ml IFN-β and a range of concentrations of LY294002 (0–50 μM), and the level of apoptosis was determined by measuring the loss of retention of DiOC6 by cells. Data are the mean ± SD of three separate experiments. b, Neutrophils were treated with 800 IU/ml of IFN-β for up to 10 min or with 500 nM fMLP for 5 min before lysis and Western blotting for the presence of phosphorylated, active PKB (pPKB). Equal loading was confirmed by probing gels for total PKB content. The blot shown is representative of three experiments performed. c, Neutrophils were treated with 800 IU/ml of IFN-β or 500 nM fMLP for 30 s before lysis and estimation of PKB activity using a commercial in vitro peptide substrate phosphotransferase assay.

Alternative PI3K targets are activated by IFN-β

PKC is an isoenzyme family of lipid-dependent kinases that are differentially involved in the regulation of apoptosis (16) and could act as downstream targets of PI3K. The classical PKC isoenzyme PKC-α has been particularly associated with anti-apoptotic signaling (17). In contrast, PKC-δ has been proposed to have predominantly proapoptotic actions, as it is cleaved and activated by caspase 3 (21, 22, 23, 24). In addition, PKC has been implicated in mediating the actions of type 1 IFN in other cell systems (32), and PKC-δ has been identified as the serine kinase responsible for type 1 IFN-mediated phosphorylation of STAT1 (33). The possible involvement of PKC isoenzymes was determined initially by assessing the translocation of PKC isoenzymes to the cell membrane fraction, which is an indicator of enzyme activation. Neutrophils express only some of the 11 PKC isoenzymes, with only PKC-α, PKC-β, PKC-δ, and PKC-ζ present at significant levels. Fig. 4⇓a shows that IFN-β treatment induced an increase in PKC-δ associated with the plasma membrane fraction. IFN-β treatment had no effect on the location of other PKC isoenzymes present in neutrophils, namely PKC-α, PKC-ζ (Fig. 4⇓a), and PKC-β (data not shown). The phorbol ester PMA was used as a positive marker of PKC activation and produced a complete translocation of PKC-α and -δ to the membrane fraction (Fig. 4⇓a). PKC-ζ was also translocated by PMA, but this is an indirect effect, as this atypical PKC is not phorbol ester responsive. The requirement for PKC-δ activation in the anti-apoptotic effects of IFN-β was investigated using PKC-selective inhibitors. The lack of involvement of the classical PKC isoenzymes PKC-α and PKC-β was confirmed by the use of Go6976, a selective inhibitor of the classical PKC isoenzymes at the concentration used (Fig. 4⇓b), which had no effect on inhibition of neutrophil apoptosis by IFN-β. In contrast, the PKC-δ-selective inhibitor rottlerin was able to inhibit the effects of IFN-β on neutrophil apoptosis in a concentration-dependent manner (Fig. 4⇓c).

FIGURE 4.
FIGURE 4.

IFN-β activates PKC-δ. a, Neutrophils were incubated with medium alone, 10 nM PMA for 10 min, or 800 IU/ml IFN-β for 30 min before lysis and differential centrifugation to isolate the cytosol (C) and cell membrane (M) fractions of the cell, which were analyzed by Western blotting, and blots were probed with Abs to PKC-α, -δ, or -ζ. The blot shown is representative of three experiments performed. Densitometric analysis of cytosol and membrane fractions was performed for the three experiments, and band intensity was expressed as membrane/cytosol (left panel). The data shown are the mean ± SD. Neutrophils were cultured for 20 h in medium containing 800 IU/ml IFN-β in the absence (□) or the presence (▪) of 50 nM Go6976 (b) or rottlerin (c; 5–10 μM). Data are the mean ± SD of three separate experiments.

Role of the NF-κB signaling pathway in IFN-β signaling

One potential downstream target of PKC-δ and PI3K that has been implicated in survival signaling and has been shown recently to be activated by type 1 IFN (34) is NF-κB. To determine the involvement of this pathway, an inhibitor approach was used initially. SN50, which inhibits translocation of NF-κB to the nucleus, blocked IFN-β-mediated inhibition of neutrophil apoptosis (Fig. 5⇓a), suggesting a role for NF-κB in the antiapoptotic actions of IFN-β. Treatment of neutrophils with cycloheximide showed that inhibition of protein synthesis also blocked the inhibition of neutrophil apoptosis by IFN-β (Fig. 5⇓b), indicating that the effects of IFN-β were both protein synthesis and NF-κB dependent.

FIGURE 5.
FIGURE 5.

IFN-β activates NF-κB. a, Neutrophils were cultured for 20 h in medium alone (□) or medium containing 800 IU/ml IFN-β (▪) in the absence (control) or the presence of the NF-κB inhibitor SN50 (100 μg/ml), and the level of apoptosis was determined. b, Neutrophils were cultured for 20 h in the absence (□) or the presence (▪) of 800 IU/ml IFN-β with or without 1 μg/ml cycloheximide, and the level of apoptosis was determined. For a and b, data are the mean ± SD of three separate experiments. c, Neutrophils were cultured with LPS as a positive control or with 800 IU/ml IFN-β in the absence or the presence of 20 μM LY294002 or 10 μM rottlerin for 20 min. Ten micrograms of nuclear extract was used in an EMSA. DNA-protein complexes were separated on polyacrylamide gels, and binding of radiolabeled oligonucleotide was detected by autoradiography. The image shown is representative of three similar experiments performed.

An EMSA to detect NF-κB activation revealed that the transcription factor was activated following treatment with IFN-β (Fig. 5⇑c). LPS treatment was used as a positive control (Fig. 5⇑c), and the specificity of NF-κB binding in the EMSA was confirmed by incubation with a 100-fold excess of cold oligonucleotide, which completely removed the NF-κB band (data not shown). Furthermore, the activation of NF-κB by IFN-β was blocked by LY294002 and rottlerin (Fig. 5⇑c), indicating that activation of NF-κB occurred downstream of PI3K and PKC-δ.

Discussion

We show here that IFN-β, which is able to inhibit T cell apoptosis (10), was also able to significantly delay neutrophil apoptosis at physiologically relevant concentrations of IFN-β. IFN-β also maintained the response of neutrophils to fMLP, thus delaying the functional senescence of neutrophils aged in vitro. This study set out to determine the molecular basis of inhibition of apoptosis by IFN-β. IFN-β signaling is coupled to the activation of PI3K (12, 35), and we have shown here that the antiapoptotic effects of IFN-β were PI3K dependent. The downstream targets of this proximal signaling kinase in mediating the antiapoptotic effects of IFN-β were also determined and were found to involve PKC-δ and NF-κB.

PKB is widely reported to be a key mediator of PI3K-mediated cell survival induced by a variety of growth factors. PKB activity has been shown to prevent apoptosis induced by cytokine and growth factor withdrawal, cellular stress, chemotherapeutic agents, and irradiation (reviewed in Ref.36). However, we (10) and others (37) have reported discordance between activation of the PI3K signaling pathway and cytokine-mediated cell survival. In neutrophils, GM-CSF and IFN-β both delay neutrophil apoptosis in a PI3K-dependent manner, but only GM-CSF activated PKB (10). FMLP and insulin both activated PKB in neutrophils, but did not delay apoptosis (10). The ability of cytokines IL-3, IL-4, stem cell factor, and GM-CSF to activate PKB did not correlate with their ability to promote cell survival in factor-dependent hemopoietic cell lines (37). These data suggested that an alternative signaling pathway was operating downstream of PI3K to induce IFN-β-mediated survival of neutrophils. One potential target already identified as playing a role in mediating the effects of type 1 IFN (32, 33) was PKC. We had already shown that the novel PKC isoenzyme PKC-δ was translocated to the nucleus and proteolytically activated by caspase 3 during spontaneous apoptosis in T cells and neutrophils (25, 36). Kufe’s group (38) reported that nuclear translocation of PKC-δ during UV irradiation-induced apoptosis was induced by tyrosine phosphorylation mediated by c-Abl. Whether c-Abl or other tyrosine kinases cause nuclear translocation of PKC-δ during T cell or neutrophil apoptosis remains to be established. However, IFN-β was able to induce relocation of PKC-δ away from the nucleus during T cell apoptosis, preventing its activation by caspase 3 and its involvement in the nuclear phase of apoptosis (25). This rather indirect action of IFN-β was unlikely to fully explain its antiapoptotic effect in neutrophils, as it was targeted at an event downstream of caspase 3 activation, and IFN-β was able to block events upstream of caspase 3 activation and mitochondrial membrane permeability transition. The data reported here show that IFN-β induced translocation of PKC-δ to the cell membrane, indicative of enzyme activation, suggesting that when PKC-δ is activated via lipid second messengers it has an antiapoptotic role.

The activation of PKC-δ via PI3K has been shown to induce NF-κB activation and cell survival (26), and we show here that PKC-δ and NF-κB were also activated by IFN-β. Moreover, inhibition of activation of either PI3K or PKC-δ completely blocked activation of NF-κB and ablated the survival effects of IFN-β. Engagement of the type 1 IFN receptor leads to the phosphorylation of Stat1 on both tyrosine and serine residues (Ser727), which is required for the transcriptional activity of Stat1. The serine kinase responsible for the phosphorylation of Ser727 has been identified as PKC-δ (33), supporting a role for the latter in transcriptional activation by type 1 IFNs. In addition, activation of NF-κB by IFN-α and IFN-β has been reported by Pfeffer’s group (34), who suggested that activation of this pathway is able to mediate survival signals in apposition to various apoptotic stimuli. PKC-δ may therefore link PI3K and NF-κB signaling pathways downstream of type 1 IFN receptor ligation (39). NF-κB regulates the expression of several antiapoptotic genes and has been proposed to play a role in regulating neutrophil apoptosis (40, 41), although the relevant gene targets have not been identified. NF-κB-regulated genes include some members of the Bcl-2 family of proteins, including Bcl-x (42), as well as proinflammatory cytokines (43). The current literature suggests that neutrophil apoptosis is initiated at the mitochondria, with mitochondrial permeability transition induced by the insertion of Bax into the mitochondrial membrane (44). IFN-β was able to prevent mitochondrial permeability transition, and therefore the up-regulation of Bcl-2 family proteins would appear to be a likely antiapoptotic effector mechanism for type 1 IFN. Moreover, we have shown previously that Bcl-2 and Bcl-xL were induced by IFN-β in T cells (8). However, neutrophils do not express Bcl-2 or Bcl-xL (45, 46). That de novo protein synthesis is required for the rescue of neutrophils from apoptosis has been shown here for IFN-β and has been reported for other survival factors. GM-CSF, which inhibits neutrophil apoptosis through the PI3K/PKB pathway (10), has been shown to up-regulate the Bcl-2 family protein Mcl-1, which declines in expression as neutrophils age in vitro (46, 47). Mcl-1 expression is regulated not by NF-κB, but by CREB and STAT transcription factors (48, 49), although another Bcl-2 family protein expressed in neutrophils, A1, is regulated by NF-κB (50). An alternative target for NF-κB in the type 1 IFN-mediated inhibition of neutrophil apoptosis would be proinflammatory cytokines, several of which are able to delay neutrophil apoptosis (4, 51). Ongoing studies using multiplex technology are now aimed at determining whether IFN-β induces the secretion of NF-κB-regulated cytokines that could mediate the inhibition of neutrophil apoptosis.

In summary, inhibition of neutrophil apoptosis by type 1 IFNs can lead to the inappropriate survival of functional neutrophils at sites of inflammation. The findings of this study show that the antiapoptotic actions of IFN-β depend upon cross-talk between three major signaling pathways, namely PI3K, PKC-δ, and NF-κB. NF-κB is a key modulator of neutrophil apoptosis, and our data show that both PI3K and PKC-δ activities are necessary for its activation by IFN-β. This cross-talk should be susceptible to pharmacological intervention, offering novel potential therapeutic approaches to the treatment of RA.

Acknowledgments

We thank H. Chahal for technical assistance.

Footnotes

  • ↵1 This work was supported by the Arthritis Research Campaign (to D.S.T.), the Biotechnology and Biological Sciences Research Council (to R.C.), and the United Birmingham Hospitals Endowment Fund (to K.W.).

  • ↵2 K.W. and D.S.-T. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. Janet M. Lord, MedicalResearch Council Center for Immune Regulation, Birmingham University Medical School, Birmingham, U.K. B15 2TT. E-mail address: j.m.lord{at}bham.ac.uk

  • ↵4 Abbreviations used in this paper: RA, rheumatoid arthritis; AMC, 7-amino-4-methyl-coumarin; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C.

  • Received March 12, 2003.
  • Accepted May 16, 2003.

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