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. 2013 Sep 16;11(1):105–106. doi: 10.1038/cmi.2013.36

SOCS3 dictates the transition of divergent time-phased events in granulocyte TNF-α signaling

Jasmeet Kaur Chhabra 1, Brajadulal Chattopadhyay 2, Bhola Nath Paul 1
PMCID: PMC4076751  PMID: 24037182

Abstract

Tumor-necrosis factor-α (TNF-α)-driven nuclear factor-κB (NF-κB) activation and apoptosis are opposing pathways; the growing recognition of these conflicting roles of TNF-α is perplexing. Here, we show that inflammation and apoptosis are time-phased events following TNF-α signaling and that emergence of suppressor of cytokine signaling 3 (SOCS3) expression limits the ongoing NF-κB activation and promotes apoptosis; further, we suggest an altered view of how inflammatory diseases are initiated and sustained. In vitro, TNF-α (50 ng/ml) induced granulocyte SOCS3 protein, inhibited nuclear accumulation of the p65NF-κB subunit and enhanced apoptosis, as shown by DNA laddering, annexin V positivity, and overexpression of caspase-3 and Bax in the late phase, whereas the early phase was marked by NF-κB activation. Conversely, SOCS3 knockdown by small interfering RNA (siRNA) inhibited granulocyte apoptosis and enhanced nuclear accumulation of p65 and 5′ lipooxygenase expression in the late phase of TNF-α signaling. As apoptosis is associated with SOCS3 abundance, we suggest that these divergent TNF-α-driven events are time-phased, interconnected, opposing control mechanisms and one of the central features through which the immune system resolves pulmonary inflammation. Dysregulation may initiate mucosal inflammation, thus changing the landscape of asthma therapy.

Keywords: apoptosis, granulocytes, lung inflammation, NF-κB, TNF-α, SOCS3

INTRODUCTION

Tumor-necrosis factor-α (TNF-α) is a chemoattractant for neutrophils and eosinophils,1,2 which are known to play important roles in asthma pathogenesis. TNF-α is initially produced as a biologically active 26-kD membrane-anchored precursor protein3 that is subsequently cleaved, principally by TNF-α-converting enzyme,4 to release the 17-kD free protein. These proteins form into biologically active homotrimers,5 which act on the ubiquitously expressed TNF-α receptors I and II (p55 and p75 or TNFRI and TNFRII).6 The receptor–ligand interaction causes intracellular signaling without internalization of the complex and phosphorylation of nuclear factor-κB (NF-κB) to activate the p50–p65 subunit, and finally, it interacts with the DNA chromatin structure to increase the transcription of pro-inflammatory genes, such as IL-8, IL-6 and TNF-α itself. The response to TNF activation is balanced by shedding the extracellular domains of the TNF-α receptors.6

Agents that block TNF-α suppress inflammation, slow disease progression, and in some cases, induce remission in patients with rheumatoid arthritis, ankylosing spondylitis, psoriasis, Crohn's disease and refractive asthma.7,8 In addition, several clinical trials of anti-TNF-α therapy have been conducted with Infliximab, a chimeric mouse/human monoclonal antibody, Etanercept, a soluble fusion protein combining two p75 TNF receptors with an Fc fragment of human IgG1, and Adalimumab, a fully human monoclonal antibody.9 As a result of the inconsistent outcomes of these trials, the impact(s) of TNF-α on granulocyte functions, in the context of asthma, remain unclear. In contrast, a dysregulated TNF-α response has been implicated in asthma, particularly refractory asthma, which is a disease state that is unresponsive to treatment with inhaled corticosteroids, and it represents 5%–10% of patients with asthma. Treatment options are limited, and there is a large unmet clinical need for additional therapies.9 Relevant to asthma, TNF-α is chemoattractant for neutrophils and eosinophils.2 Additionally, TNF-α increases the cytotoxic effect of eosinophils on endothelial cells,10 activates T cells to release cytokine11 and increases epithelial expression of adhesion molecules such as ICAM-1 and VCAM-112 with roles in the conduction of T cells to the lung and in the subsequent development of airway hyper-responsiveness.13 Recruitment of neutrophils,14 induction of glucocorticoid resistance15 and myocyte proliferation,16 and stimulation of fibroblast growth and maturation to myofibroblasts by promoting TGF-α expression17,18 are properties relevant to refractory asthma.

Exposure of cells to TNF or lipopolysaccharides can activate NF-κB.19 TNF-α is one of the most potent NF-κB activators, causing rapid phosphorylation of inhibitor κBs (IκB) at two sites within their N-terminal regulatory domain.20 NF-κB is composed of homo- and heterodimers of five members of the Rel family including NF-κB1 (p50), NFκB2 (p52), RelA (p65), RelB and c-Rel (Rel). Heterodimerization and homodimerization of NF-κB proteins exhibit differential binding specificities and include p50/RelA, p50/c-Rel, p52/c-Rel, p65/c-Rel, RelA/RelA, p50/p50, p52/p52, RelB/p50 and RelB/p52.21 These dimers are sequestered in the cytosol of unstimulated cells via non-covalent interactions with a class of inhibitor proteins called IκBs.22 NF-κB is an important regulator in cell fate decisions, such as programmed cell death and proliferation control, and is also critical in inflammation,19 an opposing pathway, but the clinical consequences of TNF-induced NF-κB activation in granulocytes remain elusive. TNF-α is a regulatory component of airway inflammation and tissue-specific apoptosis.19,23 A defect in apoptosis may contribute to the chronic tissue eosinophilia associated with asthma.24,25 The inflammation- and apoptosis-inducing properties of TNF-α26 appear perplexing.

The growing recognition of the multiple conflicting roles of TNF-α drove us to hypothesize that TNF-driven inflammation and apoptosis are time-phased events and that the negative regulator of cytokine signaling, suppressor of cytokine signaling 3 (SOCS3), may provide an intrinsic mechanism to limit ongoing NF-κB-induced inflammation and promote apoptosis. This is primarily because SOCS3 proteins can compete with the docking of signaling substrates to receptors, interfere with Janus tyrosine kinase activity, and target proteins for proteasomal degradation.27 Considering that there is very little information regarding the phasing of inflammatory and apoptotic events by TNF-α and the interplay between SOCS3 and NF-κB in granulocytes, in this report, we demonstrate that the TNF-driven immune response comprises multiple opposing reactions, the initial phase being dominated by NF-κB-mediated inflammation and the latter by SOCS3 induction and apoptosis. By inducing granulocyte apoptosis, TNF-α limits the prolonged accumulation of inflammatory granulocytes and suppresses the release of hazardous substances in the lung that may otherwise have implications for asthma pathophysiology.

MATERIALS AND METHODS

Isolation of agranulocytes and granulocytes

HIV- and hepatitis B- and C-negative blood from healthy volunteers was procured from a local Blood Bank in Lucknow, India. Granulocytes were isolated from the blood by a rapid single-step method involving centrifugation of blood over Ficoll-Hypaque. Fresh blood supplemented with EDTA was diluted twofold with cold phosphate-buffered saline (PBS) (pH 7.2) containing 2 mM EDTA, layered over Ficoll-Hypaque and centrifuged at 400g for 30 min at 20 °C in a swinging bucket rotor with no brake. Granulocytes were harvested from the lower leukocyte band, below the Ficoll/plasma interface. The cells were incubated with a lysis solution for 10 min at room temperature and then centrifuged at 300g for 10 min at 20 °C to remove contaminating red blood cells. To remove platelets, cell pellets were resuspended separately in PBS and centrifuged 3 times at 200g for 10 min at 20 °C, and the supernatants were discarded. The viability of the cells was examined by the trypan blue dye exclusion test and found to be routinely >97%. The purity of the granulocytes was assessed on Giemsa-stained leukocyte smears. Agranulocyte contamination of the granulocytes was 3%±1.2%.

Cells and cell culture

Granulocytes were cultured in RPMI 1640 (Sigma-Aldrich, Inc., St Louis, MO, USA). Media were supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified atmosphere with 5% carbon dioxide at 37 °C. Cells were resuspended at 1×106 cells/ml and allowed to rest overnight before stimulation. The cells were cultured in the presence of recombinant human TNF-α (Sigma-Aldrich) for various lengths of time.

Isolation of RNA and mRNA quantitation

Total cellular RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) as per the manufacturer's instructions. The mRNA expression profile was evaluated by standard reverse transcription polymerase chain reaction (RT-PCR) using the OneStep RT-PCR kit following the protocol published elsewhere28,29 and quantitative studies by real-time reverse transcription polymerase chain reaction (RT2-PCR) using the QuantiTect SYBR green RT-PCR kit (Qiagen).29 For reverse transcription, reactions were maintained at 50 °C for 30 min. RT-PCR was conducted with an initial activation step at 95 °C for 15 min, followed by 40 cycles of denaturation at 94 °C for 15 s, annealing for 30 s at 5 °C below the TM of each primer set and extension for 30 s at 72 °C. Relative quantifications were performed in a LightCycler Real-Time PCR Machine (Roche Diagnostics GmbH, Mannheim, Germany) using the LightCycler 480 relative quantification software (version 1.2.0625). β-actin was used as reference control. The amounts of the detected target gene products were normalized to that of β-actin before statistical analysis. The primers (Sigma-Aldrich, Inc.) used for RT-PCR were as follows: 5′ lipooxygenase (5′LO), forward (5′-GCTCAGGAGACCACGCAT-3′) and reverse (5′-CGTCCATCCCTCA GGACAAC-3′); NF-κB, forward (5′-TTCACTTTTCCAGACGCCCT-3′) and reverse (5′-ATTGTCTCTTACCGTCCCG-3′); caspase-3, forward (5′-GTGCTTCTGAGCCATGG-3′) and reverse (5′-AGTCCAGTTCTGTACCACGG-3′); Bax, forward (5′-GGTGCCTCAGGA TGCGT-3′) and reverse (5′-CTCCCGGAGGAAGTCCAATG-3′); SOCS3, forward (5′-ATGGTCACCCACAGCAAGTT-3′) and reverse (5′-CTTAAAGCGG GGCATCGTACTG-3′); β-actin, forward (5′-AGGCTGTGCTGTCCCTCT-3′) and reverse (5′- TCCGGTGA GGAGGATGCG-3′).

Western blot/dot blot analysis

Western blot analyses were performed in cytosolic and nuclear fractions of TNF-treated and untreated granulocytes. Cytosolic and nuclear fractions of granulocytes were prepared using Qproteome Nuclear Protein Kit (Qiagen) as per the manufacturer's instructions. The total protein concentration was measured by a colorimetric method using the Total Protein Kit (Spinreact, Girona, Spain). 3T3 lysates containing 176 µg, 88 µg and 44 µg of proteins and 100 µg of nuclear and cytosolic granulocyte protein samples (in a 10 µl volume) were mixed with an equal volume of 2× Laemmli sample buffer, boiled at 100 °C for 5 min, briefly centrifuged at 16 000g and loaded into the wells of a stacking gel. Proteins were separated in an SDS–PAGE (12%) resolving gel for 90–120 min at 100 V and thereafter transferred to a nitrocellulose membrane (0.2 µm; Whatman GmbH, Dassel, Germany) for 2 h at 4 °C in wet conditions using 1× Tris-glycine buffer containing methanol to a final concentration of 20%. Milk protein solution (5%) was used for blocking the free sites on the membrane for all proteins except the phosphorylated proteins, in which case 4% bovine serum albumin (Cohn fraction V) solution was used. Blocking was performed by incubating for 1 h at 4 °C under agitation. For the dot blot, 25 µg of cytosolic or nuclear fractions was hand blotted on the membranes. After blocking, the membranes were probed with rabbit polyclonal antibodies against the different proteins, i.e., p65 (A) subunit of NF-κB (sc-109), IκBα (sc-203), SOCS3 (sc-9023), histone H2A (sc-10807) and β-actin (sc-81178) (Santa Cruz Biotechnology, Inc., CA, USA), at dilutions ranging from 1∶1000 to 1∶3000. Phospho-IκBα (Ser 32) rabbit mAb # 2859 and phospho-NF-κB p65 (Ser 536) rabbit mAb # 3033 (Cell Signaling Technology, Inc., Danvers, MA, USA) were used at a 1∶1000 dilution. Goat anti- rabbit IgG antibody conjugated to horseradish peroxidase (Santa Cruz Biotech) was used as a secondary probe at a dilution of (1∶3500), and using a chemiluminescent peroxidase substrate (Sigma-Aldrich), blots were visualized using a VersaDoc Gel imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). To detect β-actin, goat anti-mouse IgG antibody conjugated to horseradish peroxidase (Sigma, St. Louis, MO, USA) at a dilution of 1∶3500 was used.

Flow cytometric analysis of apoptosis

TNF-α-stimulated cells (50 ng/ml) were assayed for apoptosis by flow cytometry at different time points. Granulocytes were labeled with annexin V (AV) (BD Pharmingen, San Diego, CA, USA) and propidium iodide (PI) (Sigma-Aldrich), following the manufacturer's staining protocol. In brief, cells were washed twice with cold PBS, resuspended in 100 µl of 1× binding buffer (0.1 M HEPES (pH 7.5), 1.4 M NaCl, 25 mM CaCl2) and incubated with annexin V-FITC (5 µl) and 10 µl of PI (50 µg/ml) for 15 min at room temperature in the dark. Thereafter, an additional 400 µl of binding buffer was added, and the samples analyzed in a flow cytometer (Partec, Münster, Germany) within 1 h. Samples were gated using forward scatter and side scatter plots. Controls were included for compensation, and quadrants were set for AV−PI− cells (viable), AV+PI− cells (early apoptotic), AV+PI+ cells (late apoptotic/necrotic) and AV−PI+ cells (dead). Each subpopulation was expressed as a percentage of the total population of cells. Annexin V binds to phosphatidylserine and, when conjugated to a fluorochrome, detects apoptotic cells expressing phosphatidylserine on the reversed membrane surface. PI was used as a counterstain to detect necrotic and late apoptotic cells.

Degradation of DNA (DNA laddering)

To determine the extent of DNA fragmentation and apoptosis, DNA laddering assays were performed by gel electrophoresis. Cells were harvested and centrifuged at 500g for 5 min and washed twice with PBS. The cell pellet was lysed in 400 µl of lysis buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.1% SDS, 0.2% Triton X-100 and proteinase K (0.1 mg/mL) at 50 °C for 16 h, followed by 1 h incubation with RNaseA (50 mg/ml) at 50 °C. DNA was extracted once with phenol/chloroform/isoamyl alcohol (25∶24∶1) and twice with chloroform∶isoamyl alcohol (24∶1). The aqueous phase was precipitated with 2 volumes of 100% ethanol at −20 °C overnight. The precipitates were rinsed with 70% ethanol, air dried, dissolved in TE buffer (10 mM Tris-HCl (pH 7.5) and EDTA (1 mM)) and electrophoresed on a 0.8% agarose gel with loading buffer. Gels were stained with ethidium bromide (5 mg/ml) for 30 min, destained, and photographed under a ultraviolet gel documentation system (Syngene International Ltd., Los Altos, CA, USA).

Cytological evaluation of TNF-α-induced granulocyte apoptosis

Granulocytes were resuspended in 20% fetal bovine serum at a concentration of 2×106 cells/ml, and an aliquot (∼10 µl) was smeared on 18-mm microscopic cover glass and submerged in one well of a six-well tissue culture plate containing media and TNF-α (50 ng/ml) after 5–10 s. Cells were then incubated at 37 °C for 0.5, 4 and 8 h. Cells adhering to the cover glass were stained with Giemsa and photographed under a Nikon Eclipse E1000 microscope at ×60 magnification.

In vitro inhibition of SOCS3 gene expression by small interfering RNA (siRNA)

SOCS3 siRNA was transfected into granulocytes 24 h prior to culture with TNF-α. A pool of 3 SOCS3 siRNA duplexes (Santa Cruz Biotech) was diluted in 100 µl of siRNA transfection medium (sc-36868) without serum or antibiotics, and 8 µl of transfection reagent (sc-29528) was diluted in 100 µl of transfection medium (Santa Cruz Biotech) and added to the granulocytes. After 24 h, cells were washed three times and cultured in RPMI 1640 in the presence of TNF-α for 4 h, and RNA was extracted. RT-PCR, RT2-PCR and flow cytometry were performed. The scramble sequence control cells were cultured with transfection reagent containing a scrambled sequence: 5′-UUCUCCGAACGUGUCACG Udtdt-3′ (Qiagen).

Statistical analysis

The data shown represent the mean values (±s.d.). Student's t-test was used to compare the activities of different treatments.

RESULTS

TNF-α-driven subcellular distribution of p65 and SOCS3 in granulocytes at 4 h

Enhanced granulocyte survival is a feature of the pathogenesis of persistent airway inflammation in asthma,30 and increased numbers of granulocytes have been observed in the sputum of both asthma and COPD patients.31 We elucidate the subcellular distribution of the p65 NF-κB subunit in granulocytes treated with TNF-α (50 ng/ml) for 4 h in vitro. 3T3 cell lysates were used as reference controls. Experiments were performed at this dose, as the physiological (15 pg/ml) and pathogenic (5 ng/ml) levels of TNF-α did not elicit measurable changes in many in vitro experimental studies32 or in our initial experiments with these doses of TNF-α (Supplementary Figure 1). The granulocyte nuclear p65 level (Supplementary Figure 1a), TNF-α-induced NF-κB expression (Supplementary Figure 1b), and apoptosis (Supplementary Figure 1c) were unaffected at the physiological and pathogenic doses of TNF-α. Cell viability based on trypan blue dye exclusion further revealed high mortality in granulocytes treated with 50 ng/ml TNF-α at 4 and 24 h. In contrast, mortality was low in granulocytes treated with doses less than 50 ng/ml at all time points (Supplementary Table I). Due to the absence of perceptible cell cytotoxicity at the physiological and pathological doses of TNF-α and a substantial degree of granulocyte mortality (∼53%) at a dose of 50 ng/mL, the remaining in vitro experiments were performed at a TNF-α concentration of 50 ng/ml.

In granulocytes treated with TNF-α for 4 h, the p65 NF-κB subunit was predominantly localized in the cytoplasm compared to the basal level in granulocytes (untreated cells) (Figure 1a and b). A significant quantity of IκBα protein was also observed in the cytoplasm of TNF-α-treated granulocytes (Figure 1a and c). In contrast, the nuclear level of p65 was similar to the basal level in TNF-treated granulocytes, whereas the IκB levels were elevated (Figure 1a–c). 3T3 fibroblast lysates were used as reference bands in the blots. Phosphorylated p65 (phos-p65) and phosphorylated IκBα (phos-IκBα) levels in the nuclear and cytosolic fractions were also monitored at 4 h. A low level of phos-p65 was observed in both the nuclear and cytosolic fractions of TNF-treated granulocytes (Figure 1e and f) in comparison to the basal level (TNF-α-untreated group). Because the p65 NF-κB subunit controls many genes involved in inflammation, a low level of nuclear p65 in 4 h TNF-treated granulocytes may result in reduced inflammation, and in this context, we presume that SOCS3 provides an intrinsic mechanism to limit TNF-induced NF-κB nuclear accumulation and subsequent inflammation. The SOCS3 protein expression profiles in the cytoplasm and nucleus of TNF-treated granulocytes were similar to the p65 expression profiles in the respective compartments. The cytosolic SOCS3 level in TNF-treated granulocytes was higher than the basal level (Figure 1a and d). 3T3 cell lysates were used to generate the reference SOCS3 bands; however, the band intensity did not correspond clearly to the loaded concentration. However, the positions of the bands were similar to those of granulocyte SOCS3 (Figure 1a and c). The increased level of SOCS3 protein in the cytoplasm of TNF-treated granulocytes may be due to stabilization of SOCS3 mRNA,33 SOCS3 gene expression34 or both. TNF-α is a potent inhibitor of IL-6-mediated STAT3 activation in human monocyte-derived macrophages, rat liver macrophages, and RAW 264.7 mouse macrophages and has been shown to induce the expression of SOCS3 mRNA in each of these types of macrophages.34 We studied the expression profile of TNF-α-induced SOCS3 mRNA using standard RT-PCR (Figure 2a) and RT2-PCR (Figure 2b) at several time points. The 700-bp PCR product represents the SOCS3 amplicon. Elevated levels of SOCS3 mRNA were observed in granulocytes both at 1 and 4 h post-TNF treatment compared to the basal level, indicating that TNF-α can induce SOCS3 gene expression in granulocytes. However, at 4 h, SOCS3 mRNA expression was increased three- to fourfold compared to 1 h. To determine whether the loss of SOCS3 gene expression alters the p65 profile in different granulocyte compartments, we treated normal granulocytes with varying concentration of SOCS3 siRNA (50–100 pmol) in vitro and thereafter treated the cells with TNF-α (50 ng/ml) for 4 h. SOCS3 knockdown by specific siRNA inhibited the TNF-α-induced expression of SOCS3 mRNA (Figure 2c and d) and protein (Figure 2e and f) in a dose-dependent manner. A high concentration of siRNA (100 pmol) reduced >80% of granulocyte SOCS3 mRNA at 24 h post-transfection. Interestingly, silencing granulocyte SOCS3 enhanced the nuclear accumulation of the p65 NF-κB subunit (Figure 2e and g). Additionally, IκB was also observed in the granulocyte nucleus, although to a lesser extent compared to scramble sequence-transfected granulocytes (Figure 2e and h). Together, these results suggest that at 4 h, TNF-α-induced SOCS3 expression correlates with the low level of nuclear p65 NF-κB subunit localization in granulocytes.

Figure 1.

Figure 1

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Figure 1 Subcellular distribution of p65 and SOCS3 in TNF-treated granulocytes. (a) Representative western blots showing the cytosolic and nuclear distributions of p65, IκBα, SOCS3 and β-actin in 4 h TNF-α-treated granulocytes. Lanes 1–3 represent 3T3 cell lysates (positive controls), and 178, 89 and 45 µg of lysate proteins were loaded, respectively; lanes 4–7 received 100 µg of nuclear or cytosolic granulocyte proteins. Quantitative data (mean±s.d., N=6; three different experiments with two blots from each) showing the subcellular distributions of p65 NF-κB subunit in b, IκBα in c and SOCS3 in d by band intensities. (e) Representative western blot showing the cytosolic and nuclear distributions of phosphorylated p65 (phos-p65) and phosphorylated IκBα (phos-IκB) and the respective housekeeping proteins, histone H2a (H2a) and β-actin in 4-h TNF-α-treated granulocytes. (f) Quantitative data (mean±s.d., from two blots) showing the sub-cellular distributions of phos-p65 and phos-IκBα. 3T3 indicates 3T3 fibroblast lysates. IκB, inhibitor κB; NF-κB, nuclear factor κB; SOCS3, suppressor of cytokine signaling 3; TNF, tumor-necrosis factor.

Figure 2.

Figure 2

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Figure 2 Relationship of SOCS3 with NF-κB activation in late phase TNF signaling in granulocytes. (a) Gel image of RT-PCR products and (b) the graphical representation of RT-PCR data showing the mRNA expression profile of SOCS3 in 4-h TNF-α-treated granulocytes. In b, C represents calibrator, and T represents target gene expression. (c) Gel image of SOCS3 mRNA in SOCS3-silenced granulocytes treated with TNF-α for 4 h and (d) bar diagram showing % remaining gene expression in granulocytes treated with varying concentrations of SOCS3 siRNA. (e) Western blots showing protein expression profiles of cytosolic SOCS3 and β-actin and nuclear p65, IκBα and histone H2a (nuclear housekeeping protein) in 4 h TNF-treated SOCS3-silenced granulocytes. SS indicates the scramble sequence-treated group. (f–h) Quantitative data showing the protein expression profiles (mean±s.d., N=4) of SOCS3, p65 and IκBα, respectively. IκB, inhibitor κB; NF-κB, nuclear factor κB; RT-PCR, reverse transcription polymerase chain reaction; SOCS3, suppressor of cytokine signaling 3; SS, scramble sequence; TNF, tumor-necrosis factor.

Early and late phases of TNF-α signaling in granulocytes

NF-κB is a rapidly acting primary transcription factor that does not require new protein synthesis; the basal level of the molecule upon dissociation from the inhibitor molecule IκB translocates to the nucleus and functions as a transcription factor. We studied the distribution of the p65 NF-κB subunit in the nuclear and cytosolic fractions of TNF-treated granulocytes in the early phase (<1 h) of TNF signaling. This subunit is a strong activator for a wide variety of genes. Phosphorylated p65 interacts with κB DNA and co-activators such as CREB-binding protein, p300 and acetyl transferases to promote specific gene activation.35

SOCS3 plays a key role in the immune and hematopoietic systems by regulating signaling induced by specific cytokines. It functions by inhibiting the catalytic activity of the Janus kinases that initiate signaling within the cell. The crystal structure of a ternary complex between mouse SOCS3, Janus kinase 2 (kinase domain) and a fragment of the IL-6 receptor β-chain reveals that the phosphotyrosine-binding groove on the SOCS3 SH2 domain binds to the IL-6 receptor,36 a critical step in the mechanism of the SOCS3-mediated negative regulation of cytokines. This being a cytosolic event, in the present experiment, we emphasized on the status of cytosolic SOCS3. However, because the p65 NF-κB subunit finally interacts with the DNA chromatin structure to increase the transcription of pro-inflammatory genes, such as IL-8, IL-6 and TNF-α, the status of nuclear p65 was emphasized here.

Temporal studies revealed a peak nuclear accumulation of p65 at 30 min post-coculture with TNF-α (Figure 3a and b). At this time point, a faint band of SOCS3 protein was observed, indicating a low level of SOCS3 in the early phase of TNF signaling. A temporal study covering both the early and late phases of TNF-α signaling was further conducted to determine the time of SOCS3 expression following TNF-α treatment (Figure 3c and d). A significant level of SOCS3 protein was observed from 2 h onward post-TNF-α treatment. Unlike granulocyte nuclear accumulation of p65 peaking in the early phase, cytosolic SOCS3 protein abundance was observed beyond the early phase. These observations indicate that the nuclear accumulation of p65 is an early effect and that the emergence of granulocyte SOCS3 is a relatively delayed event in TNF signaling.

Figure 3.

Figure 3

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Figure 3 Early phase of TNF-α-induced accumulation of p65 and SOCS3 expression in granulocytes. A representative western blot showing p65 and SOCS3 protein expression profiles during the early phase of TNF signaling is shown in a, and the quantitative temporal graph is shown in b. (c) Representative western blot of expanded temporal profile of SOCS3 protein expression. (d) Bar diagram showing quantitative data (mean±s.d., N=4) of SOCS3 expression. SOCS3, suppressor of cytokine signaling 3; TNF, tumor-necrosis factor.

An elaborate study over the course of TNF-α signaling in granulocytes is shown by western and dot blots (Figure 4). Elevated levels of phos-p65 were observed in the nuclear fraction of granulocytes up to 1 h by western blotting (Figure 4a) and up to 2 h by dot blotting (Figur. 4C and D). In the cytoplasm, phos-p65 was also elevated at 1 and 2 h post-TNF treatment (Figure 4c and d). Phos-IκBα levels were also elevated in the nuclear fraction through 1 h post-TNF exposure in both western and dot blots (Figure 4a, e and f). The SOCS3 level in the cytoplasm was consistently high at 4 and 8 h (Figure 4b–d). In the nucleus, SOCS3 was found at the basal level at all time points (Figure 4e and f). Collectively, these results show some degree of regularity in terms of NF-κB activation and SOCS3expression, despite the random appearance of SOCS3 at 0.5 h (Figure 4b), phos-IκBα at 4 h (Figure 4a) and phos-p65 at 24 h (Figure 4a). The emerging trend within these anomalies appears to support the hypothesis that NF-κB activation is an early event and that SOCS3 expression is a delayed event following TNF-α signaling in granulocytes.

Figure 4.

Figure 4

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Figure 4 Demonstration of time phase stages of TNF-α signaling in granulocytes. Western blot showing the course of TNF-α signaling (0–8 h) in terms of phosphorylated p65 and IκBα in the granulocyte nuclear fraction (a) and SOCS3 in the cytosol (b). Histone protein (H2a) and β-actin represent housekeeping proteins in the nuclear and cytosolic fractions, respectively. Dot blot showing the time course of TNF-α signaling (0–24 h) in terms of phos-p65, phos-IκBα and SOCS3 in granulocyte nuclear (c) and cytosolic (e) fractions. (d, f) 3D ribbon plots of c and e displaying trends over time. IκB, inhibitor κB; SOCS3, suppressor of cytokine signaling 3; TNF, tumor-necrosis factor.

TNF-α induces apoptosis in granulocytes in the late phase

TNF-α-treated granulocytes, stained with Annexin V-FITC and counterstained with PI, were subjected to flow cytometric analysis to distinguish and quantitatively determine the percentage of dead, necrotic, apoptotic and viable granulocytes at different time points following in vitro exposure to TNF-α. TNF-α induced significantly more granulocyte apoptosis (AV+PI−) after 4 h and 8 h of treatment compared to the basal level of apoptosis (0 h). At the early time points (0.5, 1 and 2 h), changes in apoptosis were insignificant compared to the basal level of apoptosis (Figure 5a and b). Although a good number of dead cells were observed in the dot plots at the 1 and 2 h time points, it is not clear whether the dead cell population largely represents apoptotic granulocytes. Eukaryotic cell death can also occur due to necrosis, entosis, mitotic catastrophe, autophagy and pyroptosis. Nonetheless, based on annexin positivity, early apoptotic cells dominate largely from 4 h onward in TNF-α signaling in granulocytes. To microscopically evaluate TNF-α-induced granulocyte apoptosis, granulocytes were cultured in the presence of TNF-α (50 ng/ml) for varying time periods, smeared on cover glass, stained with Giemsa, and viewed for morphological changes. Disruption of granulocyte membranes and loss of polymorphonuclear shape were observed after 4 h of culture with TNF-α. Evidence of cell death was more prominent at 8 h post-stimulation (Figure 5c). These findings indicate that TNF-α elicits the apoptotic pathway in the late phase of signaling. A DNA degradation assay performed at various time points demonstrated DNA laddering after 8 h of culture with 50 ng/ml TNF-α (Figure 5d). At 4 h, visible DNA laddering was not observed, although significantly elevated levels of AV+PI− granulocytes were detected at this time point. Caspase-3 (Cas3) is the critical executioner of apoptosis, and granulocytes are susceptible to apoptosis.37 Hypermorphic expression of Cas3 was observed at 8 h in TNF-α-treated granulocytes, reaffirming the onset of programmed cell death (Figure 5E). Thus, apoptosis-associated annexin positivity, cell morphology (membrane damage), Cas3 expression and DNA degradation in granulocytes arise during the late phase of TNF-α signaling.

Figure 5.

Figure 5

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Figure 5 The late phase of TNF signaling is marked by granulocyte apoptosis. Representative flow cytometric analysis of TNF-α-induced human granulocyte death. The dot plot diagrams represent the typical apoptotic and necrotic cell populations detected by Annexin V-FITC and PI staining. Cells treated with 50 ng/ml TNF-α for 0, 0.5, 1, 2, 4 and 8 h are shown in a. The upper left quadrant (Q1: FITC−/PI+) shows dead cells, and the lower left quadrant (Q3: FITC−/PI−) shows viable (intact) cells, which were negative for both Annexin V-FITC and PI binding. The upper right quadrant (Q2: FITC+/PI+) shows nonviable, necrotic cells, positive for Annexin V-FITC binding and PI uptake. The lower right quadrant (Q4 FITC+/PI−) represents apoptotic cells, positive for Annexin V-FITC and negative for PI. (b) Bar diagram showing TNF-α-induced apoptosis (mean±s.d.) in granulocytes. N=5 culture data; *P<0.05. (c) Microscopic demonstration of TNF-α-induced granulocyte apoptosis. (d) Agarose gel image showing DNA fragmentation in granulocytes treated with TNF-α (50 ng/ml) after 8, 4 and 0.5 h of coculture. (e) Electrophoretic analysis of standard RT-PCR products on an agarose gel showing expression of caspase-3 and β-actin in granulocytes treated with TNF-α at different time points. M: molecular weight markers; PI, propidium iodide; RT-PCR, reverse transcription polymerase chain reaction; TNF, tumor-necrosis factor.

Role of SOCS3 in the late phase of TNF signaling

To further establish that SOCS3 initiates apoptosis in the late phase of TNF-α signaling, the expression levels of 5′LO, NF-κB, caspase-3 and Bax were measured in SOCS3-silenced granulocytes treated with TNF-α for 4 h. The expression profiles of pro-apoptotic (Cas3 and Bax) and inflammation-associated molecules (5′LO and NF-κB) were studied in TNF-α-treated SOCS3-knockdown granulocytes by RT-PCR (Figure 6a) and RT2-PCR (Figure 6b). Upregulation of 5′LO and N-FκB expression was observed in granulocytes silenced with 100 pmol of SOCS3 siRNA. Cas3 and Bax were unaffected. A significant decrease in granulocyte apoptosis was further observed in SOCS3-silenced granulocytes compared to scramble sequence-transfected cells (Figure 6c). To summarize the optimum relationship between cytoplasm SOCS3 and TNF exposure duration that favors apoptosis, a 3D surface chart was created with the apoptosis/necrosis ratio on the vertical axis, the SOCS3 siRNA transfecting dose on the horizontal axis, and the duration of TNF exposure on the depth axis (Supplementary Table 2). Knockdown of SOCS3 in granulocytes decreased granulocyte apoptosis in a dose-dependent manner, and a >200-fold increase in the apoptosis-necrosis ratio was observed in native granulocytes at 4 h (Figure 6d). These findings demonstrate that the emergence of SOCS3 in the late phase of TNF-α signaling can influence granulocytes to switch between inflammation and apoptosis.

Figure 6.

Figure 6

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Figure 6 Inflammation- and apoptosis-associated gene expression profiles in SOCS3-silenced granulocytes in the late phase of TNF signaling. SOCS3-knockdown granulocytes were stimulated with 50 ng/ml TNF-α for 4 h in vitro, and RT-PCR and RT2-PCR were performed. Graphical representations of 5′LO, NF-κB, caspase-3, Bax and β-actin mRNA expression profiles in granulocytes are shown in a and b. (c) Flow cytometric dot plots of TNF-α-mediated apoptosis of SOCS3-silenced granulocytes. (d) 3D surface chart showing the optimum combination of SOCS3 siRNA and TNF exposure duration that favors apoptosis. Apoptosis is responsible for clearance of inflamed granulocytes, whereas necrotic granulocytes release inflammatory molecules into the local environment in bulk. The former event is associated with resolution of inflammation, whereas the later promotes inflammation. As apoptosis and necrosis coexist in the granulocyte population, we prepared the chart using the apoptosis/necrosis ratio on the y-axis. 5′LO, 5′ lipooxygenase; NF-κB, nuclear factor κB; RT-PCR, reverse transcription polymerase chain reaction; RT2-PCR, real-time reverse transcription polymerase chain reaction; siRNA, small interfering RNA; SOCS3, suppressor of cytokine signaling 3; TNF, tumor-necrosis factor.

DISCUSSION

The role of TNF-α in inflammatory diseases such as asthma is not clear. Asthma is a multifactorial disease, and TNF-α is a pleiotropic cytokine that regulates multiple cellular responses, including inflammation and cell survival. It is produced by many cell types (including macrophages, monocytes, lymphocytes, keratinocytes and fibroblasts) in response to inflammation, infection, injury and other environmental challenges. A genome-wide study has reported that TNF-α is a candidate gene in asthma38 and that it upregulates 5′LO and 5′LO-associated protein. The 5′LO pathway-derived leukotrienes, including the cysteinyl leukotrienes and LTB4, have been implicated in the pathogenesis of a number of inflammatory and allergic disorders, including asthma, sepsis, acute lung injury, pulmonary fibrosis and atherosclerosis.39,40,41 Incidentally, TNF-α is a well-known activator of NF-κB, inducing inflammation, and also a recognized inducer of apoptosis, an opposing pathway to inflammation. These conflicting roles of TNF-α appear perplexing, and the present study was an attempt to resolve the issue regarding how this molecule can induce divergent self-opposing pathways and to understand the reason behind the inconsistent outcomes of trials evaluating anti-TNF agents in the treatment of asthma. In this investigation, we demonstrated that NF-κB activation and apoptosis are time-phased events of TNF-α signaling and that the emergence of SOCS3 limits the initial NF-κB activation and initiates a functionally opposing event, apoptosis. The first phase of TNF-α activity induces inflammation, demonstrated by NF-κB activation and 5′LO expression. The second phase is initiated by the emergence of SOCS3, which correlates with reduced nuclear accumulation of the active NFκB subunit and promotes apoptosis. It is possible that this is the normal innate immune response elicited during infection or xenobiotic exposure, which culminates in the secretion of TNF-α. The objective of this process is to induce the apoptosis of inflammatory cells and their clearance. With the help of these divergent events, the host maintains homeostasis by regulating signaling pathways and removing unwanted inflammatory cells from the airways. This is one of the central features of the immune system, as reactions are highly controlled and coordinated. Within the cells, there may be divergent interconnected opposing control mechanisms to contain pulmonary inflammation.

Neutrophils are short-lived cells that migrate in large numbers from the blood to inflamed tissues early in the acute inflammatory response. Upon activation, these cells produce toxic oxygen metabolites42 and release antimicrobial and tissue-damaging lytic enzymes that are stored in granules.43 Apoptosis is the key mechanism of limiting the release of the hazardous contents of neutrophils and preventing tissue damage of the neighboring areas. We demonstrated here that granulocytes were susceptible to TNF-induced apoptosis and that TNF-α, despite its pro-inflammatory label, helped to resolve inflammation by selectively preventing the accumulation of granulocytes in the subepithelial region beneath the basement membrane of the airway. The mechanism that interferes in the inhibition of the TNF-α-induced NF-κB inflammatory pathway may lead to asthma, as observed in a previous genome-wide association study where multiple single nucleotide polymorphisms in the TNFAIP3-interacting protein 1 (TNIP1) gene, which interacts with TNFAIP3 and inhibits the TNF-α-induced NF-κB inflammatory pathway, were associated with asthma.44

Alternatively, the mechanism that interferes with SOCS3 expression may also lead to the pathological manifestation of acute and chronic inflammation. SOCS3 has been reported to promote the apoptosis of differentiated mammary cells,45 and silenced SOCS3 can regulate the expression of apoptosis-associated genes via the Janus kinase/STAT3 pathway and effectively inhibit TNF-α-induced apoptosis in 3T3-L1 preadipocytes and mouse preadipocytes.46 A defect in granulocyte apoptosis may contribute to the pathophysiology of asthma, e.g., granulocyte-mediated inflammation of the airways is a key characteristic of asthma.47,48

The clinical benefits of anti-TNF-α therapies were likely minimal in asthma and Etanercept, a fusion protein of the TNF-α receptor, was likely not clinically efficacious in a 12-week study because inflammation and apoptosis are time-phased events of TNF signaling and apoptosis is the eventual event.49 Because silencing SOCS3 decreased TNF-α-induced apoptosis and because a defect in apoptosis might contribute to the chronic tissue eosinophilia associated with asthma, we believe that SOCS3 upregulation may be a better strategy for treating asthma and related inflammatory diseases, thus changing the landscape of asthma therapy. As lipopolysaccharide and TNF-α were found to induce SOCS3 expression34 and because the spontaneous secretion of TNF-α by peripheral monocytes leads to the generation of ROS and the upregulation of SOCS3,50 it will be worthwhile to determine whether these modes of SOCS3 upregulation can benefit asthmatic patients.

Acknowledgments

This is CSIR-IITR communication # 3009. JKC is a recipient of a University Grant Commissions-Senior Research Fellowship. This work was supported by CSIR, New Delhi (NWP-33). JKC was involved in the experimentation and the acquisition, analysis, and interpretation of data and searched the literature and helped in the critical revision of the manuscript for important intellectual contents; BC helped in the revision of the manuscript for intellectual contents; BNP obtained funding and was involved in conceptualizing and designing the study, interpreting the data, drafting the manuscript and supervising the study overall.

The authors declare no competing financial interests.

Footnotes

Supplementary Information accompanies the paper on Cellular & Molecular Immunology website.

Supplementary Information

Supplementary information
Click here for additional data file. (342.6KB, pdf)

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